Novel human androgen receptor alternative splice variants as biomarkers and therapeutic targets

ABSTRACT

The present invention relates to novel androgen receptor splice variants (AR3, AR4, AR4b, AR5 and AR8) and variants and fragments thereof which have a role in the progression of androgen independent prostate cancer. The invention further relates to compositions and methods which can be used to identify and treat prostate cancer based on these novel androgen receptor splice variants, as well as methods for screening agents which modulate the activity and/or expression of the androgen receptor splice variants. Vectors, host cells and recombinant methods for producing the same and transgenic animals are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional U.S. Appl. No.61/097,571, filed Sep. 17, 2008. The content of the aforesaidapplication is relied upon and incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant NumberCA106504 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns at least the fields of cell biology,molecular biology and medicine. In particular aspects, the presentinvention concerns the fields of treatment and/or prevention of prostatecancer.

2. Related Art

Prostate cancer is the most commonly diagnosed malignancy in males inthe United States and the second leading cause of male cancer mortality.In the event that surgery and/or radiation do not cure prostate cancer,systemic therapy is based on inhibiting the androgen receptor (AR). Theandrogen receptor is a steroid receptor transcription factor whichpromotes the growth and survival of normal and cancerous prostate cells.Androgen ablation is used to block the activation or activity ofandrogens initially and results in a favorable clinical response.However, prostate cancer invariably occurs in a manifestation which isfatal and is resistant to androgen ablation. This stage of prostatecancer is termed androgen refractory prostate cancer.

Prostate cancer is the second leading cause of cancer death among men inwestern countries. Patients with advanced prostate cancer initiallybenefit from androgen ablation therapy which leads to temporaryremission of the tumor due to apoptosis of androgen-sensitive tumorcells. However, the recurrence of androgen independent tumors isinevitable for most patients and renders the conventional hormonetherapy ineffective (Denmeade, S. R. et al., Nat Rev Cancer 2, 389-96(2002)). It has, therefore, become a focus of intensive study tounderstand the mechanisms underlying progression of hormone refractoryprostate cancer (Litvinov, I. V., et al., Prostate 61, 299-304 (2004);Feldman, B et al., Nat Rev Cancer 1, 34-45 (2001); and Debes, J. D. etal., N Engl J Med 351, 1488-90 (2004)). Development of new effectivetherapeutic agents is necessary for targeting hormone refractoryprostate cancer. As prostate cancer is a product of slow but continuousaccumulation of altered genes and proteins controlling several signalingpathways it thus poses a difficult task for identification of precisemarkers to predict high risk for recurrence. Validation of tumorprogression markers are an essential component for prostate cancertreatment strategies to plan for appropriate therapy based on its level,tissue distribution and the stage or extent of the disease.

Identification and characterization of the molecular mechanisms andbiological switches which propel normal prostate cells into cells in thecancerous state is critical for developing therapeutic modalities whichcan be used to prevent and/or treat male subjects predisposed toprostate cancer.

Increasing clinical findings demonstrate that a majority of androgenablation therapy-resistant prostate cancers still express AR andandrogen-dependent genes, indicating that the AR-signaling pathway isfunctional in the absence of androgens or in the presence of low levelsof androgens (Chang, C. S. et al., Science 240, 324-6 (1988); Lubahn, D.B. et al., Mol Endocrinol 2, 1265-75 (1988)). Several independentstudies also showed that AR is essential for both hormone sensitive andrecurrent hormone refractory prostate cancer (McPhaul, M. J. et al., JInvestig Dermatol Symp Proc 8, 1-5 (2003); Heinlein, C. A. et al.,Endocr Rev 25, 276-308 (2004)). The mechanisms underlyingandrogen-independent activation of AR have yet to be fully understood.Mutations and amplification of AR, alterations in protein kinases,growth factors and nuclear receptor coactivators have all been proposedto modulate AR signaling and may, therefore, play key roles in thedevelopment of androgen independence of prostate cancer (Feldman, B. etal., Nat Rev Cancer 1, 34-45 (2001); Lubahn, D. B. et al. Mol Endocrinol2, 1265-75 (1988); Kuiper, G. G. et al., J Mol Endocrinol 2, R1-4(1989)). Mutations in the ligand binding domain of AR are shown tobroaden the ligand binding profile of the mutant receptor (Lubahn, D. B.et al., Proc Natl Acad Sci USA 86, 9534-8 (1989); Libertini, S. J. etal., Cancer Res 67, 900 1-5 (2007); Simental, J. A., et al., J Biol Chem266, 5 10-8 (1991)). However, the frequency of AR mutation is generallylow and probably only accounts for less than 10% of the cases surveyed(Jenster, G. et al. Mol Endocrinol 5, 1396-404 (1991)). Upregulation ofthe enzymes involving in steroid synthesis in some recurrent prostatetumors and activation of AR via the intracrine mechanism have also beenreported (Rundlett, S. E. et al., Mol Endocrinol 4, 708-14 (1990)).However, the tissue androgen level did not correlate with clinicalprognosis. Recently, the increased AR expression level is shown toassociate with the development of resistance to anti-androgen therapy(McPhaul, M. J. et al., J Investig Dermatol Symp Proc 8, 1-5 (2003)).The cross-talk between growth factor and AR signaling pathways inprostate cancer cells has been well documented. The expression ofseveral peptide growth factors, such as EGF/TGFa, IL-6 and IGF-1, arereported to be elevated during progression to hormone refractory humanprostate cancer (Bolton, E. C. et al., Genes Dev 21, 2005-17 (2007);Wang, Q. et al., Mol Cell 27, 3 80-92 (2007); Clegg, N. et al. J SteroidBiochem Mol Biol 80, 13-23 (2002); DePrimo, S. E. et al., Genome Biol 3,RESEARCH0032 (2002); Marcelli, M. et al., J Clin Endocrinol Metab 84,3463-8 (1999); Comuzzi, B. et al., Am J Pathol 162, 233-41 (2003); Chen,C. D. et al., Nat Med 10, 33-9 (2004)). These autocrine/paracrinefactors can either induce the androgen-independent activation of ARtranscriptional activity or sensitize AR to low concentrations ofandrogens (Lubahn, D. B. et al., Mol Endocrinol 2, 1265-75 (1988);Zegarra-Moro, O. L., et al., Cancer Res 62, 1008-13 (2002); Culig, Z. etal., Endocr Relat Cancer 9, 155-70 (2002)). Several protein kinases,including MAPK, Akt/PKB, PICA and PKC, Src and Ack-1 have been shown tomodulate AR transcriptional activity by phosphorylating AR or itscoactivators such as TIF2 and SRC1 (Zegarra-Moro, O. L., et al., CancerRes 62, 1008-13 (2002); Culig, Z., et al., Endocr Relat Cancer 9, 155-70(2002); Gelmann, E. P. et al., J Clin Oncol 20, 3001-15 (2002);Mizokami, A. et al., Cancer Res 64, 765-7 1 (2004); Kurita, T. et al.,Cell Death Differ 8, 192-200 (2001); Veldscholte, J. et al., BiochemBiophys Res Commun 173, 534-40 (1990); Taplin, M.-E. et al., J ClinOncol 21, 2673-2678 (2003)). Another plausible hypothesis for activationof androgen receptor activity in the absence of hormones was proposed byTepper et al. who showed that a mutant AR identified in hormonerefractory prostate cancer cell line CWR22Rv1 contains an in-frametandem duplication of exon 3 that encodes the second zinc finger of theAR DNA-binding domain (Paez, J. G. et al., Science 304, 1497-1500(2004)). This insertional mutation renders AR susceptible to theprotease cleavage at the hinge region and generates a constitutivelyactive form around 80 kD. A recent study also showed that calpain maymediate cleavage of the AR mutant in this cell line (Yeh, S. et al.,Proc Natl Acad Sci USA 96, 5458-63 (1999)). However, the frequency ofsuch insertional mutation has only been detected in CWR22R xenograftderived cell line CWR22Rv1. It remains elusive whether it is a generalmechanism underlying androgen-independence.

We have cloned novel AR splice variants (AR3, AR4, AR4b, AR5 and AR8)from hormone refractory CWR22Rv1 xenografts. All the variants containthe intact N-terminal transactivation domain and the DNA binding domain,but lack the ligand binding domain, and therefore, are trueandrogen-independent. AR3 appears to be constitutively active inARE-mediated transcription. We confirmed the expression of these ARvariants in human prostate tumors by RT-PCR and/or immunohistochemistry.Knocking-down AR3, one of the major AR variants in hormone refractoryprostate cancer cells, attenuated androgen-independent growth in bothcell culture and xenograft models. Our data suggest that the AR variantsresulted from alternative splicing may be a novel mechanism underlyingandrogen-independence during prostate cancer progression. These novel ARvariants are not inhibited by currently available anti-androgen drugs(such as casodex) due to the lack of the ligand binding domain.Therefore new drugs targeting at these novel AR variants may potentiallybe more effective for androgen-independent prostate tumors. Furthercharacterization of these AR variants will provide new insights intomechanisms underlying androgen-independence and identify new therapeutictargets. Detection of the expression of these AR variants may bepotentially used as a prognostic marker for prostate cancer andtargeting these variants may develop more effective treatment forandrogen-independent prostate cancer.

BRIEF SUMMARY OF THE INVENTION

Example embodiments are generally directed to novel androgen receptorsplice variants designated as AR3, AR4, AR4b, AR5 and AR8. These novelandrogen receptor splice variants can play an important role in theprogression of androgen independent progression of prostate cancer.These androgen-independent receptors contain the N-terminaltransactivation domain and the DNA binding domain but lack the ligandbinding domain.

In other embodiments, these splice variants can serve as prognosticmarkers (biomarkers) to determine the degree of the disease and predictoutcome in response to hormonal therapy.

In other embodiments, these novel androgen receptor splice variantsrepresent targets for therapeutics/drugs which can be used to treathuman subjects diagnosed with prostate cancer. In particular, theseembodiments cover a variety of methodologies which can be used tomodulate these novel androgen receptors, including but not limited to:antisense technology, natural and/or synthetic compounds orpeptides/proteins, RNAi, ribozymes and antibody technology.

A first aspect of the invention provides novel, isolated androgenreceptor (AR) nucleic acids and polypeptides that are not available inthe art.

More specifically, isolated nucleic acid molecules are provided encodingAR3, AR4, AR4b, AR5 and AR8 and variants and fragments thereof. Vectors,host cells and recombinant methods for producing the same are alsoprovided.

Another aspect of the invention relates to isolated AR3, AR4, AR4b, AR5and AR8 polypeptides and variants and fragments thereof and methods fortheir production.

Another aspect of the invention relates to methods for using AR3, AR4,AR4b, AR5 and AR8 polynucleotides and polypeptides. Such uses includeassays for screening agents that modulate either the expression orprotein activity of AR3, AR4, AR4b, AR5 or AR8, among others.

Another aspect of the invention relates to diagnostic assays and kitsfor the detection of diseases associated with AR3, AR4, AR4b, AR5 or AR8expression levels or protein activity associated with neoplasticdisorders, such as androgen refractory prostate cancer.

Another aspect of the invention relates to methods for detectingandrogen refractory prostate cancer in an animal comprising assaying anandrogen receptor splice variant expression level in the animal andcomparing the androgen receptor splice variant expression level with astandard androgen receptor splice variant expression level.

Another aspect of the invention relates to pharmaceutical compositionscomprising a therapeutically effective agent which regulates expressionof AR3, AR4, AR4b, AR5 or AR8.

Another aspect of the invention relates to isolated antisense nucleicacids that are sufficiently complimentary to the nucleic acid sequenceof AR3, AR4, AR4b, AR5 or AR8 to permit hybridization under physiologicconditions, and which antisense nucleic acid inhibits expression of theandrogen receptor.

Another aspect of the invention relates to methods for alteringexpression of the androgen receptor splice variants AR3, AR4, AR4b, AR5or AR8 in a prostate cancer cell comprising delivering agents to thecell which increase or decrease the stability of mRNA coding for AR3,AR4, AR5 or AR8.

Another aspect of the invention relates to methods of treating orpreventing androgen refractory prostate cancer in a patient in needthereof, comprising administering to the patient an effective amount ofan agent that inhibits the function of an androgen receptor splicevariant selected from the group consisting of AR3, AR4, AR4b, AR5 andAR8.

Another aspect of the invention relates to methods for treating cancerin an animal comprising administering to the animal an isolated shortinterfering RNA molecule that inhibits expression of the androgenreceptor splice variant AR3, AR4, AR4b, AR5, or AR8.

Another aspect of the invention relates to methods for inhibiting theactivity of androgen splice receptor variants AR3, AR4, AR4b, AR5 or AR8in a prostate cancer cell comprising administering to said cell anantibody or functional fragment of the antibody which binds the androgenreceptor.

Another aspect of the invention relates to a screening system fordetecting compounds which bind to nucleic acids encoding AR3, AR4, AR4b,AR5 or AR8, thereby inhibiting expression of the androgen receptor.

Another aspect of the invention relates to transgenic animals or theirprogeny or parts thereof, which comprises in their genome a transgeneconstruct comprising a polynucleotide encoding an androgen receptorsplice variant selected from the group consisting of AR3, AR4, AR4b, AR5and AR8. Such transgenic animals are useful as animal models forandrogen refractory prostate cancer, among other uses.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention are herein described, by way ofnon-limiting example, with reference to the following accompanyingFigures:

FIG. 1: Cloning of novel alternative splice AR isoforms. (a) Presence ofthe short forms of AR in prostate cancer cells. Total cell lysates ofdifferent prostate cancer cells were blotted with anti-AR (441) antibody(upper) and anti-Actin antibody (lower). (b) Selective knockdown of ARlong/short forms by AR shRNAs. CWR-R1 cells were infected with thelentivirus encoding AR shRNAa, AR shRNAb, and AR shRNAc (shARa, shARband shARc) targeting different exons of AR as indicated in (d). At 48 hrpost infection, the total cell lysates were subjected to western blotwith anti-AR antibody. The same blot was stained with Coomassie Blue toshow the protein loading (right panel). (c) Presence of AR isoformtranscripts in CWR-R1 cells. Total RNA was isolated from CWR-R1 cellsand reverse transcribed. The primer derived from AR shRNAc sequence wasused to perform a rapid amplification of cDNA 3′-end (3′-RACE) with theRACE oligo-dT primer or a control primer. (d) Schematic structure of thehuman AR splice variants. The hatched cassettes stand for the crypticexons. Solid thick lines represent the transcribed sequences. (e)Expression of AR isoforms in prostate cancer cells. The relativeexpression levels of AR, AR3, AR4 and AR5 were quantified usingreal-time PCR with the isoform specific primer pair sets described inMethods (left panel). The AR level in LNCaP cells was arbitrarily setas 1. The expression levels of AR3 in LNCaP and C-8 1 cells were furtherplotted with a higher resolution. *p<0.05 (right panel). (I)Transcriptional activity of AR isoforms. COS-1 cells were transfectedwith ARR.2-luciferase reporter gene together with AR, AR3, AR4 and AR5expression vector. At 24 hr posttransfection, the cells were lysed andthe luciferase activity was measured. The data were expressed asrelative promoter activity, the changes of luciferase activity relativeto the untreated control (arbitrarily set as 1). (g) COS-1 cells weretransfected with ARR2-Luciferase reporter along with increasing doses ofAR3 or AR expressing vector. At 24 hr posttransfection, the cells weretreated with or without 10 nM DHT for 24 hr and the luciferaseactivities were then measured. (h) LNCaP cells were infected with (+) orwithout (−) the lentivirus encoding AR shRNA (shAR) as describedpreviously. At 6 hr postinfection, the cells were transfected withARR2-Luciferase reporter along with AR3 or the codon-switched wild-typeAR expressing vector (ARcs). At 24 hr posttransfection, the cells weretreated with DHT and casodex (CAS) as indicated for 24 hr before theluciferase activity was measured.

FIG. 2: Detection of AR3 isoform in hormone refractory prostate cancercells by the anti-AR3 antibody. (a) COS-1 cells were transfected withAR3 or AR expression vector. The total protein lysates wereimmunoblotted with anti-AR3 and anti-AR antibodies, respectively. (b)CWR-R1 and 22Rv1 cells were infected with lentivirus encoding AR3shRNA-1, -2 (shAR3-1, -2) or the scrambled control AR3 shRNA (shAR3-sc).At 48 hr postinfection, the cells were lysed and subjected toimmunoblotting with anti-AR (441) and anti-AR3 antibodies, respectively.The lysates of COS-i cells overexpressing AR.3 were used as a positivecontrol (first lane). (c) The lysates of CWR-R1 cells were split intothree equal aliquots and immnuoprecipitated with anti-AR3 antibody,control antibody IgG and anti-AR antibody, respectively. The resultantimmunoprecipitates and the input total cell lysates (TCL) wereimmunoblotted with anti-AR. antibody. (d) Subcellular Localization ofAR3. CWR-R1 and 22Rv1 cells were subjected to immunofluorescencestaining with anti-AR3 antibody or the control pre-immune serum. Thenucleus was visualized with DAPI staining.

FIG. 3: Increased AR3 expression in hormone refractory prostate tumorcells. (a) Total cell lysates of a panel of prostate cancer cells wereblotted with anti-AR3 and anti-Actin antibodies, respectively. (b) Thehuman prostate tissue microarrays were stained with anti-AR3 antibody.The mean score of cytoplasmic and nuclear staining of the luminal cellas well as the positive rates for cytoplasmic and nuclear staining wereshown. (Error bars indicate standard error, *p<0.01). Benign prostatetissue (B), hormone naïve (MN) and hormone refractory (HR). (c) Therepresentative fields of the tissue arrays stained with anti-AR3. Theanti-AR stained arrays were included as a control. (d) Correlation ofAR3 cytoplasmic staining with PSA recurrence after prostatectomy.

FIG. 4: AR3 promotes androgen-independent growth of prostate cancercells. LNCaP cells were infected with lentivirus encoding AR3 expressionconstruct (AR3) or the control vector. After 2-week culture inandrogen-depleted condition, the cells were visualized by Coomassie Bluestaining (a). At 48 hr post infection, the cells were injected into thecastrated SCID mice and the growth of the tumor cells were monitoredweekly. The result represent the mean tumor volume±SE (n=5 mice/group),*p<0.05 Inset, Western blots of anti-AR3 and anti-AR of the LNCaPxenograft tumor lysates (b). CWR-R1 and 22Rv1 cells were infected withlentivirus encoding AR3 shRNA-1 (shAR3) or control shRNA (shCon). After2 weeks culture in androgen-depleted condition, the cells werevisualized by Coomassie Blue staining (c). At 48 hr postinfection, thecells were injected into the castrated male nude mice. The growth of thetumors was monitored weekly. The result represents the mean tumorvolume±SE (n=5 mice/group), *p<0.05 (d). Inset, Western blots ofanti-AR3 and anti-AR of the CWR-R1 and 22Rv1 xenograft tumor lysates.

FIG. 5: AKT1 is a target gene regulated by AR3. (a) Schematicrepresentation of the AR3 and AR regulated genes. (b) The effects of AR3shRNA on the transcription of endogenous AKT1. The CWR-R1 cells wereinfected with lentivirus encoding AR3 shRNA-1 (shAR3), AR shRNA (shAR)or AR3 shRNA scrambled control (shAR3-sc). At 48 hr post infection, thetotal RNA was isolated and reverse transcribed. The relative expressionlevels of AKT1 transcripts compared with the vector (Control) wasquantified by real-time PCR (*p<0.05). (c) CWR-R1 cells were infectedwith the lentivirus encoding shAR3, shAR and shAR3-sc. 1IPr-1 cells wereinfected with the lentivirus encoding AR, AR3 or the vector control. At48 hr post infection, the cells were lysed and the protein levels ofAKT1, Actin, AR3 and AR were detected by immunoblotting. The levels ofAKT1 from the immunoblots were normalized by calculating the ratios ofAKT1/actin. The changes in fold compared to the control were shown(bottom). (d) The lysates of LNCaP and 22Rv1 xenograft tumors from FIGS.4 a and 4 b were subjected to immunoblotting as described in (c). (e)CWR-R1 and 22Rv1 cells were treated with or without DHT (10 nM) for 1hr. The binding of AR3 and AR to the different putative ARE sites (P1,P2 and P3) of human AKT1 gene was analyzed by the ChIP assay An AREbinding site at the PSA enhancer region (PSA) was used as a positivecontrol for AR. PCR products from input (1), immunoprecipitation withanti-AR3 antibody (2), anti-AR antibody (3) or the control antibody (4),were resolved on agarose gels.

FIG. 6. The postulated role of AR3 in prostate cancer cells.

FIG. 7. Expression of AR, AR3, AR4, and AR5 in human normal andmalignant prostate tissues.

FIG. 8. Increased AR3 expression in hormone refractory prostate tumorxenografts.

FIG. 9. Specificity of the anti-AR3 antibody.

FIG. 10. IHC staining of AR3 in human prostate tissues.

FIG. 11. AR3 full-length cDNA sequence (SEQ ID NO:1) and proteinsequence (SEQ ID NO:5).

FIG. 12. Putative AR4 full length cDNA sequence (SEQ ID NO:2) andprotein sequence (SEQ ID NO:6)

FIG. 13. Putative AR5 full length cDNA sequence (SEQ ID NO:3) andprotein sequence (SEQ ID NO:7).

FIG. 14. The panels depict information relating to the generation andanalysis of an AR3 transgenic (AR3Tg) mouse.

FIG. 15. Histological analysis of tissue sections revealed that theAR3Tg displayed extensive hyperplasia in all three lobes.

FIG. 16. AR3Tg exhibited an increase in Ki67-positive cells.

FIG. 17. The number of p63-positive cells is significantly increased inAR3Tg prostates.

FIG. 18. Elevated MAPK and Akt pathways in AR3Tg prostate determined byWestern blot with anti-phosphoMAPK and pan anti-phospho AKT-substratesantibodies, respectively.

FIG. 19. A partial list of genes regulated by AR3 in mouse prostate.

FIG. 20. The level of mature TGF-β monomers (doublet suggesting at leasttwo members of TGF-β family may be processed in prostate) wasdramatically elevated in AR3Tg prostate compared to the WT control. As aresult, both SMAD1 and SMAD3 phosphorylation is elevated.

FIG. 21. The number of Sca-1 and α6 integrin double positive cells wasincreased at least two fold to 35% in AR3Tg prostates compared to 16% inthe WT littermate controls.

FIG. 22. The prostatic cells from the AR3Tg mouse appeared to form morespheres than those from the WT littermate controls and AR3 transgene ishighly expressed in the basal compartment of the sphere. 6-week-oldAR3Tg or WT control prostates were dissected and minced, followed bydigestion with collagenase for 90 min. Dissociated cells were then berun through a BD FACSVantage Cell Sorter to remove cell doublets andsmall clusters. The isolated single cells were counted and suspended inMatrigel/PrGEM(1:1), then seeded in triplicate at final densities of1250, 2500, 5000 and 10,000 cells per well in 12-well plates. At 14-daypost-plating, prostatic spheres with a diameter greater than 40 um werecounted and photographed (Top). The prostatic spheres were fixed andembedded in paraffin and sectioned with the microtome at 3 μm thickness.IHC staining with antibody for AR3 were performed (Bottom).

FIG. 23. IHC screening performed on multi-tumor tissues arrays revealedthat AR3 appeared to be detected in many types of tumors, includingTestis Seminom, Testis Germ cell tumor, Lung Carcinoid tumor, LungAdenocarcinoma, Breast Ductal Carcinoma, Breast Invasive LobularCarcinoma, Basal Cell Carcinoma and Pancreas Adenocarcinoma. TS: TestisSeminoma; TG: Testis Germ cell tumor; LT: Lung Carcinoid tumor; LA: LungAdenocarcinoma; BDC: Breast Ductal Carcinoma; BLC: Breast InvasiveLobular Carcinoma; BCC: Basal Cell Carcinoma; PA: PancreasAdenocarcinoma.

FIG. 24. Association of AR and AR splicing isoforms with mitochondria.(A) COS-1 cells were transfected with AR4 expression construct. At 24 hpost transfection, cells were labeled with mitotracker dye (Invitrogen)for 30 min at 37° C. to show the mitochondria (red) and followed withthe immunofluorescence staining with anti-AR (green). (B) CWR-R1 cellswere infected with AR shRNAs. At 48 hr post infection, the cytoplasmic(Cyto) and mitochondrial (Mito) fractions of the CWR-R1 cells wereprepared by using a mitochondria isolation kit (Pierce) and subjected towestern-blot with anti-AR and anti-Bcl-xL. The latter was served as amitochondria marker.

FIG. 25. Novel AR splicing variant AR8 (FIG. 25A) and its nucleotidesequence is shown in FIG. 25B (SEQ ID NO:4). Schematic structure of thehuman AR splice variants. The hatched cassettes stand for the crypticexons. Solid thick lines represent the transcribed exon sequences.

FIG. 26. AR8 amino acid sequence (SEQ ID NO:8). AR8 is primarily presenton the plasma membrane when overexpressed (FIG. 26B). Identification oftwo Cysteine residues (C558 & 569) in the C-terminal unique sequence aspotential palmitoylation sites (underlined in FIG. 26A). Substitution ofthese two Cys with Ala residues dramatically diminished plasma membranetargeting of AR8 (FIG. 26B).

FIG. 27. The real-time PCR analysis revealed that AR8 expression iselevated in hormone resistant CWR22R xenografts (HR) compared to hormonesensitive counterpart (HS) (FIG. 27A) and AR8 level is also increased inhormone resistant LNCaP derivatives C4-2 and C4-2B compared to hormonesensitive parental LNCaP cells (FIG. 27B). Total RNA was isolated fromhormone sensitive (HS), hormone resistant CWR22 xenografts, or prostatecancer cell lines, and then subjected to reverse transcription-PCR. Theprimers used to amplify AR8 are (sense5′-CTACTCCGGACCTTACGGGGACATGCG-3′(SEQ ID NO:31) and antisense5′-CTTTCTTCGGGTATTTCGCATGTC-3′ (SEQ ID NO:32)). The total cell lysatesfrom these cells were subjected to immunoblotting with anti-AR (rightpanel). The position of AR8 is marked by an arrow.

FIG. 28. Hormone-resistant prostate cancer cell lines C4-2, 22Rv1 andCWR-R1 treated with the lentivirus encoding the shRNA specific for AR8exhibit attenuated growth (FIG. 28A). This is accompanied with thereduced activity of Akt, MAPK and Src kinases (FIG. 28B). Prostatecancer cells were infected with the lenti-virus encoding shRNA specificfor AR8 (shAR8, target sequence: CTCATTATCAGGTCTATCA (SEQ ID NO:30)).After 2 weeks culture in androgen-depleted condition, the cells werevisualized by coomassie blue staining.

CWR-R1 cells were treated with the control or shAR8 for 48 hrs and celllysates were subjected to immunoblotting with the indicated antibodies.The position of AR8 is marked by an arrow.

FIG. 29. AR8 knock-down inhibits proliferation (FIG. 29A) and increasesof apoptosis in CWR-R1 cells (FIG. 29B). CWR-R1 cells were plated oncoverslips and infected with lenti-virus encoding shAR8 or the vectorcontrol in androgen-depleted medium. At 48 h post-infection, the cellproliferation was evaluated by the ClickiT™ EdU Assay (Invitrogen).CWR-R1 cells were infected with lenti-virus encoding specific shAR8 orthe vector control in androgen-depleted medium. At 48 h post-infection,the apoptosis was detected by TUNEL assays.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention which, together with the drawings and thefollowing examples, serve to explain the principles of the invention.These embodiments describe in sufficient detail to enable those skilledin the art to practice the invention, and it is understood that otherembodiments may be utilized, and that structural, biological, andchemical changes may be made without departing from the spirit and scopeof the present invention. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods, devices, and materials nowdescribed.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Current Protocols in Molecular Biology(Ausubel et. al., eds. John Wiley & Sons, N.Y. and supplements thereto),Current Protocols in Immunology (Coligan et al., eds., John Wiley StSons, N.Y. and supplements thereto), Current Protocols in Pharmacology(Enna et al., eds. John Wiley & Sons, N.Y. and supplements thereto) andRemington: The Science and Practice of Pharmacy (Lippincott Williams &Wilicins, 2Vt edition (2005)) for example.

As discussed above and herein, the present invention relates to novelalternatively spliced variants of human androgen receptor as biomarkersand therapeutic targets. The novel splice variants of the androgenreceptor (AR), AR3, AR4, and AR5 and AR8 are herein described.

As used herein, unless otherwise indicated by a specific nucleotide SEQID NO, “AR3,” “AR4,” “AR4b,” “AR5,” and “AR8” also include transcriptsthat arise through the use of alternative polyadenylation signals.

AR4b (SEQ ID NOS:36-38) differs from AR4 in that it lacks a threonineresidue in the carboxy-terminal region as compared with AR4 (SEQ ID NO:2and 6), and is likely a product resulting from usage of alternativesplice donor or acceptor sequences.

These novel AR splice variants are expressed in hormone refractoryprostate cancer cells and may contribute to their androgen independence.

These AR variants contain the intact N-terminal transactivation domain,and the DNA binding domain but lack the ligand binding domain. AR3 isone of the major splice variants expressed in human prostate tissues.AR3 is constitutively active and its transcriptional activity is notregulated by androgens or antiandrogens. The data collected wouldindicate that AR3 is significantly upregulated during prostate cancerprogression and the AR3 expression level is correlated with the risk oftumor recurrence after a prostatectomy. Thus, AR3 has a putativeimportant role in the development of androgen-independence of prostatecancer. AR4 comprises two transcripts of different size, depending ondifferential usage of a 3′ polyadenylation signal. The longer version isindicated by SEQ ID NO:2 and the shorter version is indicated by SEQ IDNO:35. AR4b also gives rise to transcripts using the samepolyadenylation signals, and is indicated by SEQ ID NO:36 and 38.

Accordingly, certain embodiments of the present invention relate tovarious methods for identifying potential cancerous prostate tissuesusing the spliced androgen receptor variants as biomarkers.Additionally, certain embodiments of the present invention relate tovarious therapeutic modalities targeted to the spliced androgen receptorvariants.

Certain other embodiments of the present invention relate to variousmethods of diagnosis, prophylactic treatment and therapeutic treatmentof prostate cancer predicated on the features of these novel androgenreceptor splice variants.

Providing a therapy or “treating” refers to indicia of success in thetreatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement,remission, diminishing of symptoms of making the injury, pathology orcondition more tolerable to the patient, slowing the rate ofdegeneration or decline, making the final point of degeneration lessdebilitating, or improving a patient's physical or mental well-being.Those in need of treatment include those already with the disease(prostate cancer) as well as those prone to have the disorder those inwhom the disorder is to be prevented. Preferred subject for treatmentinclude animals, most preferably mammalian species, such as humans anddomestic animals such as dogs, cats, and the like, subject to diseaseand other pathological conditions. A “patient” refers to a subject,preferably mammalian (including human).

Protein Activity or Biological Activity of the Protein: Theseexpressions refer to the metabolic or physiologic function of theandrogen receptor splice variant proteins including similar activitiesor improved activities or these activities with decreased undesirableside-effects. Also included are antigenic and immunogenic activities ofsaid androgen receptor splice variant protein. Among the physiologicalor metabolic activities of said protein is the transcription of androgenreceptor responsive target genes, binding of coactivators, DNA or othertranscription factors and repressors. Androgen independenttranscriptional activation is also included.

Androgen receptor splice variant polynucleotides: This term refers to apolynucleotide containing a nucleotide sequence that encodes androgenreceptor splice variant polypeptides or fragments thereof, or variantsthat encodes an androgen receptor splice variant polypeptide or fragmentthereof or variant, wherein said nucleotide sequence has at least 90%identity to a nucleotide sequence encoding the polypeptide of SEQ IDNOS:5-8 or 37 or a corresponding fragment thereof, or which hassufficient identity to a nucleotide sequence contained in SEQ IDNOS:1-4, 35, 36 and 38.

Androgen receptor splice variant polypeptides: This term refers topolypeptides with amino acid sequences sufficiently similar to theandrogen receptor splice variant protein sequences in SEQ ID NOS:5-8 or37 and wherein at least one biological activity of the protein isexhibited.

I. Pharmaceutical Compositions and Methods

Certain embodiments of the present invention relate to pharmaceuticalcompositions comprising one or more therapeutic agents, and methods ofadministering a therapeutically effective amount of one or moretherapeutic agents, which are capable of prophylactic and/or therapeutictreatment of prostate cancer and related conditions. The term“therapeutic agent” refers to any pharmaceutically acceptable acid,salt, ester, derivative, a stereoisomer, pro-drug or mixtures ofstereoisomers of a therapeutic agent or to the therapeutic agent itself.Pharmaceutically acceptable acids, salts, esters, derivatives,stereoisomers, pro-drugs, and mixtures of therapeutic agents may also beused in the methods and compositions of the present invention. Thetherapeutic agents can include agents which downregulate thetranscriptional level and/or protein level of the androgen receptorsplice variants including AR3, AR4, AR4b, AR5 and AR8, agents whichmodulate the interaction between the androgen receptor splice variantsincluding AR3, AR4, AR4b, AR5 and AR8 with their co-factors, such ascoactivator proteins or DNA response elements, and agents which modulateposttranslational modification of the androgen receptor splice variantsincluding AR3, AR4, AR4b, AR5 and AR8.

The pharmaceutical compositions can be formulated according to knownmethods for preparing pharmaceutically acceptable useful compositions,and may include a pharmaceutically acceptable carrier. The carrier maybe liquid, solid, or semi-solid for example. Formulations are describedin a number of sources which are well known to those of skill in theart. The physical and/or chemical characteristics of compositions of theinventions may be modified or optimized according to skill in the art,depending on the mode of administration and the particular disease ororder to be treated. The compositions may be in any suitable form,depending on the desired method of administration, and may be providedin unit dosage form, a sealed container, or as part of a kit, which mayinclude instructions for use and/or a plurality of unit dosage forms.

The term “pharmaceutically acceptable” used herein refers to thosemodifications of the parent compound (acids, salts, ester, etc.) that donot significantly or adversely affect the pharmaceutical properties ofthe parent compound. For example, exemplary pharmaceutically acceptablesalts administrable by means of the compositions of this inventioninclude chloride, bromide, iodide, hydrochloride, acetate, nitrate,stearate, palmoate, phosphate, and sulfate salts. Exemplary techniquesfor producing pharmaceutically acceptable derivatives include forexample, methylation, halogenation, acetylation, esterification andhydroxylation.

The term “therapeutically effective amount” means the total amount ofeach active component of the pharmaceutical composition or method thatis sufficient to show meaningful patient benefits, i.e, a decrease inthe prostate tumor size or metastatic potential of the tumor, anincrease in patient survival time, sensitization of patients to othertherapeutic agents including but not limited to chemotherapy andhormonal therapy.

The pharmaceutical composition may be adapted for administration by anyappropriate route, for example by the oral, rectal, nasal, topical,vaginal or parenteral routes. Other routes, e.g., intra-articular, mayalso be used. Such compositions may be prepared by any known method, forexample by admixing the active ingredient with the carrier(s) orexcipient(s) under sterile conditions.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; as powders orgranules, as solutions, syrups or suspensions. Suitable excipients fortablets or hard gelatine capsules include lactose, maize starch, orderivative thereof, stearic acids or salts thereof. Suitable excipientsfor use with soft gelatine capsules include for example vegetable oils,waxes, fats, semi-solid, or liquid polyols etc. For the preparation ofsolutions and syrups, excipients which may be used include for examplewater, polyols, and sugars. For the preparation of suspension, oils(e.g., vegetable oils) may be used to provide oil-in-water orwater-in-oil suspensions. In certain situations, delayed release orenteric-coated preparations may be advantageous, for example to decreasegastric residence time and thereby reduce degradation of thepharmaceutical composition en route to the lower GI tract.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or enemas. Pharmaceutical compositionsadapted for nasal administration wherein the carrier is a solid includea coarse powder having a particle size ranging from about 20 to about500 microns. Suitable compositions wherein the carrier is a liquid foradministration as a nasal spray or as nasal drops, include aqueous oroil solution of the active ingredient. Pharmaceutical compositionsadapted for administration by inhalation include fine particle dusts ormists which may be generated by means of various types of metered dosepressurized aerosols, nebulizer or insufflators.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols, or oils. When formulated inan ointment, the therapeutic agent may be employed with either aparafinninic or a water-miscible ointment base. Pharmaceuticalcompositions adapted from topical administration to the eye include eyedrops wherein the therapeutic agent is dissolved or suspended in asuitable carrier, especially an aqueous solvent. Pharmaceuticalcompositions adapted for topical administration in the mouth includelozenges, pastilles, and mouth washes. Pharmaceutical compositionsadapted for transdermal administration may be presented as discretepatches intended to remain in intimate contact with the epidermis of therecipient for a prolonged period of time. Pharmaceutical compositionsadapted for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats, and solutes which renderthe formulation substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients which may beused for injectable solutions include water, alcohols, polyols,glycerine and vegetable oils, for example. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in freeze-dried conditionsrequiring only the addition of a sterile liquid immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules, and tablets. The pharmaceuticalcompositions may contain preserving agents, solubilizing agents,stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants,salts, buffers, antioxidants, etc.

The administration of the compositions of the present invention may befor a “prophylactic” or “therapeutic” purpose, or alternatively can beused for diagnostic purposes. The compositions of the present inventionare said to be administered for a “therapeutic” purpose if the amountadministered is physiologically significant to provide a therapy for anactual manifestation of the disease. When provided therapeutically, thecompound is preferably provided at (or shortly after) the identificationof a symptom of actual disease. The therapeutic administration of thecompound serves to attenuate the severity of such disease or to reverseits progress. The compositions of the present invention are said to beadministered to provide a therapy for a potential disease or condition.When provided prophylactically, the compound is preferably provided inadvance of any symptom thereof. The prophylactic administration of thecompound serves to prevent or attenuate any subsequent advance of thedisease.

II. Polynucleotides of the Invention

In some embodiments, the invention provides polynucleotides encodingandrogen receptor splice variant polypeptides having the amino acidsequence set out in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:37 and variants and fragments thereof.

A particular nucleotide sequence encoding an androgen receptor splicevariant polypeptide may be identical over its entire length to thecoding sequence in SEQ ID NOs:1, 2, 3, 4, 35, 36, or 38. Alternatively,a particular nucleotide sequence encoding a androgen receptor splicevariant polypeptide may be an alternate form of SEQ ID NOs:1, 2, 3, 4,35, 36, or 38 due to degeneracy in the genetic code or variation incodon usage encoding the polypeptides of SEQ ID NOs:5, 6, 7, 8 or 37. Insome embodiments, the polynucleotides of the invention contain anucleotide sequence that is highly identical, at least 90% identical,with a nucleotide sequence encoding an androgen receptor splice variantpolypeptide or at least 90% identical with the encoding nucleotidesequence set forth in SEQ ID NOs:1, 2, 3, 4, 35, 36, or 38.

When a polynucleotide of the invention is used for the recombinantproduction of an androgen receptor splice variant polypeptide, thepolynucleotide may include the coding sequence for the full-lengthpolypeptide or a fragment thereof, by itself; the coding sequence forthe full-length polypeptide or fragment in reading frame with othercoding sequences, such as those encoding a leader or secretory sequence,a pre-, or pro or prepro-protein sequence, or other fusion peptideportions. For example, a marker sequence that facilitates purificationof the fused polypeptide can be encoded. In certain embodiments of thisaspect of the invention, the marker sequence is a hexa-histidine peptide(SEQ ID NO:34), as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc Natl Acad Sci USA 86:821-824 (1989), orit may be the HA tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson, I., et al., Cell 37:767, 1984).The polynucleotide may also contain non-coding 5′ and 3′ sequences, suchas transcribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Embodiments of the invention include isolated nucleic acid moleculescomprising a polynucleotide having a nucleotide sequence at least 90%identical, and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% 99% or 100% identical to (a) a nucleotide sequence encoding anandrogen receptor splice variant polypeptide having the amino acidsequence in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ IDNO:37; or (b) a nucleotide sequence complementary to the nucleotidesequences in (a).

Conventional means utilizing known computer programs such as the BestFitprogram (Wisconsin Sequence Analysis Package, Version 10 for Unix,Genetics Computer Group, University Research Park, 575 Science Drive,Madison, Wis. 53711) may be utilized to determine if a particularnucleic acid molecule is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to any one of the nucleotide sequences shownin SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:35, SEQID NO:36, or SEQ ID NO:38.

In some embodiments, the polynucleotides encoding an androgen receptorsplice variant polypeptide have an amino acid sequence of the androgenreceptor splice variant polypeptides of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8 or SEQ ID NO:37, in which several, 1, 1-2, 1-3, 1-5 or5-10 amino acid residues are substituted, deleted or added, in anycombination.

In some embodiments, the polynucleotides are at least 90% identical overtheir entire length to a polynucleotide encoding an androgen receptorsplice variant polypeptide having the amino acid sequence set out in SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:37, andpolynucleotides which are complementary to such polynucleotides. In someembodiments, the polynucleotides are at least 95% identical over theirentire length, at least 97% identical, at least 98% identical, or atleast 99% identical.

The present invention is further directed to fragments of SEQ ID NOs:1,2, 3, 4, 35, 36 and 38. A fragment may be defined to be at least about15 nt, and more preferably at least about 20 nt, still more preferablyat least about 30 nt, and even more preferably, at least about 40 nt inlength. Such fragments are useful as diagnostic probes and primers asdiscussed herein and can be incorporated into detection kits to detectthe androgen receptor splice variants in biological samples. Of courselarger DNA fragments are also useful according to the present invention,as are fragments corresponding to most, if not all, of the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:35, SEQ ID NO:36 or SEQ ID NO:38. In some embodiments, the fragmentscan be at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 510, 520, 530, 540, 550, 560,570, 580, 590, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800,820, 840, 860, 880, 900, 920, 940, 960, 980 or 1000 nucleotides.

The present invention further relates to polynucleotides that hybridizeto the above-described sequences. In this regard, the present inventionespecially relates to polynucleotides that hybridize under stringentconditions to the above-described polynucleotides. As herein used, theterm “stringent conditions” means hybridization will occur only if thereis at least 90% and preferably at least 95% identity and more preferablyat least 97% identity between the sequences.

Furthermore, a major consideration associated with hybridizationanalysis of DNA or RNA sequences is the degree of relatedness the probehas with the sequences present in the specimen under study. This isimportant with a blotting technique (e.g., Southern or Northern Blot),since a moderate degree of sequence homology under nonstringentconditions of hybridization can yield a strong signal even though theprobe and sequences in the sample represent non-homologous genes.

The particular hybridization technique is not essential to theinvention, any technique commonly used in the art is within the scope ofthe present invention. Typical probe technology is described in U.S.Pat. No. 4,358,535 to Falkow et al., incorporated by reference herein.For example, hybridization can be carried out in a solution containing6×SSC (10×SSC: 1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0),5×Denhardt's (1×Denhardt's: 0.2% bovine serum albumin, 0.2%polyvinylpyrrolidone, 0.02% Ficoll 400), 10 mM EDTA, 0.5% SDS and about10⁷ cpm of nick-translated DNA for 16 hours at 65° C. Additionally, ifhybridization is to an immobilized nucleic acid, a washing step may beutilized wherein probe binding to polynucleotides of low homology, ornonspecific binding of the probe, may be removed. For example, astringent wash step may involve a buffer of 0.2.times.SSC and 0.5% SDSat a temperature of 65° C.

Additional information related to hybridization technology and, moreparticularly, the stringency of hybridization and washing conditions maybe found in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989), which is incorporated herein by reference.

Polynucleotides of the invention which are sufficiently identical to anucleotide sequences contained in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:38 may be used ashybridization probes for cDNA and genomic DNA, to isolate full-lengthcDNAs and/or genomic clones encoding androgen receptor splice variantproteins and to isolate cDNA and genomic clones of other DNA that has ahigh sequence similarity to the androgen receptor splice variants. Suchhybridization techniques are known to those of skill in the art.Typically, these nucleotide sequences are at least about 90% identical,preferably at least about 95% identical, more preferably at least about97%, 98% or 99% identical to that of the reference. The probes generallywill comprise at least 15 nucleotides. Preferably, such probes will haveat least 30 nucleotides and may have at least 50 nucleotides. In someembodiments, the probes will range between 30 and 50 nucleotides. Insome embodiments, the probes will be at least about 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 620, 640, 660, 680,700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960,980 or 1000 nucleotides.

In other embodiments, the invention provides polynucleotide sequencethat has promoter activity, operatively linked, in a transcriptionalunit, to a DNA sequence encoding a protein of interest. In oneembodiment, the DNA sequence encodes a protein of interest selected fromthe group consisting of SEQ ID NO:5, 6, 7, 8 or 37 and variants andfragments thereof. In some embodiments, the DNA sequence encodes apolypeptide fragment or variant of SEQ ID NO:5, 6, 7, 8 or 37 thatpossesses wild-type protein activity. In other embodiments, the DNAsequence encodes a polypeptide fragment or variant of SEQ ID NO:5, 6, 7,8 or 37 that is a null mutant, or a dominant negative mutant thatinhibits endogenous androgen receptor splice variant activity. In someembodiments, the promoter is tissue specific for the prostate. In someembodiments, the promoter is constitutively active, and is not selectiveto a particular tissue. In other embodiments, the promoter is inducible,using methods known in the art.

III. Vectors, Host Cells, and Recombinant Expression

The present invention also relates to vectors that comprise apolynucleotide of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells. A great variety of expression systems can beused, including DNA or RNA vectors.

In other embodiments, this invention provides an isolated nucleic acidmolecule comprising an androgen receptor splice variant operably linkedto a heterologous promoter. This invention further provides an isolatednucleic acid molecule comprising and androgen receptor splice variantoperably linked to a heterologous promoter, wherein said isolatednucleic acid molecule is capable of expressing an androgen receptorsplice variant polypeptide when used to transform an appropriate hostcell.

IV. Polypeptides of the Invention

The androgen receptor splice variant polypeptides of the presentinvention include the polypeptide of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, and SEQ ID NO:37 as well as polypeptides andfragments which have activity and have at least 90% identity to thepolypeptide of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQID NO:37. Dominant negative mutants and null mutants of SEQ ID NOS:5-8or 37 are also included. In some embodiments, the polypeptides have atleast 96%, 97% or 98% identity to the polypeptide of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:37. In some embodiments, thepolypeptides have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identity to the polypeptide of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8 or SEQ ID NO:37.

The androgen receptor splice variant polypeptides may be a part of alarger protein such as a fusion protein. It is often advantageous toinclude additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purificationsuch as multiple histidine residues, or additional sequence forstability during recombinant production.

Biologically active fragments of the androgen receptor splice variantpolypeptides are also included in the invention. A fragment is apolypeptide having an amino acid sequence that entirely is the same aspart but not all of the amino acid sequence of one of the aforementionedandrogen receptor splice variant polypeptides. As with androgen receptorsplice variant polypeptides, fragments may be “free-standing,” orcomprised within a larger polypeptide of which they form a part orregion, most preferably as a single continuous region. In the context ofthis invention, a fragment may constitute from about 10 contiguous aminoacids identified in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8or SEQ ID NO:37. In some embodiments, the fragment is about 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240 or 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490 500, 510, 520, 530, 540 contiguous amino acids or moreidentified in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQID NO:37.

In some embodiments the fragments include, for example, truncationpolypeptides having the amino acid sequence of androgen receptor splicevariant polypeptides, except for deletion of a continuous series ofresidues that includes the amino terminus, or a continuous series ofresidues that includes the carboxyl terminus or deletion of twocontinuous series of residues, one including the amino terminus and oneincluding the carboxyl terminus. In some embodiments, fragments arecharacterized by structural or functional attributes such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, high antigenic indexregions, functional domains such as the N-terminal transactivationdomain or the DNA binding domain. Biologically active fragments arethose that mediate protein activity, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Also included are those that are antigenic or immunogenic inan animal, especially in a human.

Thus, the polypeptides of the invention include polypeptides having anamino acid sequence at least 90% identical to that of SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:37 or fragments thereofwith at least 90% identity to the corresponding fragment of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:37, all of whichretain the biological activity of the androgen receptor splice variantprotein, including antigenic activity. Included in this group arevariants of the defined sequence and fragment. In some embodiments, thevariants are those that vary from the reference by conservative aminoacid substitutions, i.e., those that substitute a residue with anotherof like characteristics. Typical substitutions are among Ala, Val, Leuand Ile; among Ser and Thr; among the acidic residues Asp and Glu; amongAsn and Gln; and among the basic residues Lys and Arg, or aromaticresidues Phe and Tyr. In some embodiments, the polypeptides are variantsin which several, 5 to 10, 1 to 5, or 1 to 2 amino acids aresubstituted, deleted, or added in any combination.

The androgen receptor splice variant polypeptides of the invention canbe prepared in any suitable manner. Such polypeptides include isolatednaturally occurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art.

V. Genetic Screening and Diagnostic Assays

As a means to detect or diagnose neoplastic disorders, such as prostatecancer or androgen refractory prostate cancer, differences in the mRNAexpression levels (or cDNA) or protein levels or sequence differences ofthe androgen receptor splice variants between affected and unaffectedindividuals can be determined. Increased expression of the splicevariants in prostate cells is indicative of the presence of androgenrefractory prostate cancer.

This invention also relates to the use of androgen receptor splicevariant polynucleotides or antibodies reactive specifically against thesplice variants for use as diagnostic reagents. Detection of alteredmRNA (or cDNA) or protein levels of the androgen receptor splicevariants will provide a diagnostic tool that can add to or define adiagnosis of a disease or susceptibility to a disease which results fromaltered expression of the androgen receptor splice variants. Thedetection of normal or altered expression levels of the androgenreceptor splice variants will direct the medical practitioner to set anappropriate course of treatment for the patient.

Nucleic acids for diagnosis may be obtained, for example, from a biopsyof cells from the prostate. In some embodiments, bodily fluids, e.g.,urine, are obtained from the patient are used to detect elevated levelsof expression. Alterations in expression level can be assayed bycomparison to a standard or control expression level of androgenreceptor splice variant. RNA may be used directly for detection or maybe converted to cDNA and amplified enzymatically by using PCR or otheramplification techniques prior to analysis.

The diagnostic assays offer a process for diagnosing or determining asusceptibility to neoplastic disorders through detection of alteredexpression levels or mutations in one or more androgen receptor splicevariants by the methods described. Neoplastic disorders, such asandrogen refractory prostate cancer, may be diagnosed by methods thatdetermine an abnormally increased level of androgen receptor splicevariant polypeptide or androgen receptor splice variant mRNA in a samplederived from a subject. Decreased or increased expression may bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides; for example, RT-PCR, RNaseprotection, Northern blotting, array analysis, and other hybridizationmethods may be utilized. Assay techniques that may be used to determinethe level of a protein, such as an androgen receptor splice variantprotein, in a sample derived from a host are well known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western blot analysis and ELISA assays.

Additionally, methods are provided for detecting or determining asusceptibility of an individual to neoplastic disorders, such asprostate cancer or androgen refractory prostate cancer, comprising (a)assaying the androgen receptor splice variant expression level in ananimal such as cells or body fluid; and (b) comparing said androgenreceptor splice variant expression level with a standard androgenreceptor splice variant expression level whereby an increase in saidandrogen receptor splice variant expression level over said standard isindicative of a neoplastic disorder or an increased susceptibility to aneoplastic disorder, such as, for example, androgen refractory prostatecancer. In some embodiments, the animal is a mammal, preferably a human.In some embodiments, the androgen receptor splice variant expressionlevel is AR3 (e.g., SEQ ID NO:1), AR4 (e.g., SEQ ID NOS:2 or 35), AR4b(e.g., SEQ ID NO:36 or 38), AR5 (e.g., SEQ ID NO:3) and/or AR8 (e.g.,SEQ ID NO:4). The androgen receptor splice variants can be assayedindividually or in combination, e.g., as a panel of biomarkers toindicate the presence of androgen refractory prostate cancer. Otherprostate cancer markers, such as PCA3, can also be simultaneouslyassayed, in accordance with the methods of the present invention.

VI. Androgen Receptor Splice Variant Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them may also be used as immunogens to produceantibodies immunospecific for the androgen receptor splice variantpolypeptides. By “immunospecific” is meant that the antibodies haveaffinities for the polypeptides of the invention that are substantiallygreater in their affinities for related polypeptides such as theanalogous proteins of the prior art.

Antibodies generated against the androgen receptor splice variantpolypeptides can be obtained by administering the polypeptides orepitope bearing fragments, analogs or cells to an animal, preferably anonhuman, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature 256:495-497(1975)), the trioma technique, the human B-cell hybridoma technique(Kozbor, et al., Immunology Today 4:72 (1983)) and the EBV-hybridomatechnique (Cole, et al., Monoclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc., (1985)).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) may also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies maybe employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against androgen receptor splice variant polypeptides mayalso be employed to treat neoplastic disorders, such as prostate canceror androgen refractory prostate cancer to a patient in need thereof.

In some embodiments, the invention is directed to antibodies thatrecognize AR3, AR4, AR4b, AR5 or AR8. In some embodiments, theantibodies are monoclonal. In some embodiments, the antibodies arehumanized.

VII. Agonist and Antagonist Screening

In some embodiments, the ability of antagonists and agonists of androgenreceptor splice variant proteins to interfere or enhance the activity ofthe androgen receptor splice variant proteins can be evaluated withcells containing the androgen receptor splice variant. In someembodiments of the invention, an assay for androgen receptor splicevariant activity in cells can be used to determine the functionality ofthe androgen receptor splice variant protein in the presence of an agentwhich may act as antagonist or agonist, and thus, agents that interfereor enhance the activity of androgen receptor splice variants areidentified.

In some embodiments, the androgen receptor splice variants of thepresent invention are employed in a screening process for compoundswhich bind one of the proteins and which enhance (agonists) or inhibit(antagonists) the transcriptional activation activity of one of thepolypeptides of the present invention. Thus, polypeptides of theinvention may also be used to assess the binding of molecular substratesand ligands in, for example, cells, cell-free preparations, chemicallibraries, and natural product mixtures. These substrates and ligandsmay be natural substrates and ligands or may be structural or functionalmimetics. Antagonists of the androgen receptor splice variants areparticularly advantageous and can be used in methods as therapeuticagents in the treatment of prostate cancer, for example, androgenrefractory prostate cancer in mammals in need of treatment.

By “agonist” is intended naturally occurring and/or synthetic compoundscapable of enhancing transcriptional activity mediated by the androgenreceptor splice variants of the invention, or other biologicalactivities of the protein(s). By “antagonist” is intended naturallyoccurring and/or synthetic compounds capable of inhibitingtranscriptional activity mediated by the androgen receptor splicevariants of the invention, or other biological activities of theprotein(s).

In some embodiments, the screening procedures involve producingappropriate cells which express the polypeptide(s) of the presentinvention. Such cells include cells from mammals, yeast, Drosophila orE. coli. In some embodiments, the cells express the polypeptideendogenously. In other embodiments, the cells have been transfected orengineered to express the polypeptide. In some embodiments, cellsexpressing the protein (or extracts or purified preparations from cells)are contacted with a test compound to observe stimulation or inhibitionof a functional response. In some embodiments, one or more androgenreceptor response element(s) (ARE) are linked to a reporter gene andactivation or inhibition of the reporter gene is assayed. In someembodiments, the expression level of an endogenous androgen receptortarget gene is assayed.

In some embodiments, assays test binding of a candidate compound to theandrogen receptor splice variants or assays involving competition with alabeled competitor. In some embodiments, inhibitors of activation can betested in the presence of an agonist and the effect on activation by theagonist in the presence of the candidate compound is observed.

Examples of androgen receptor splice variant protein agonists orantagonists include antibodies, peptides, carbohydrates, vitaminderivatives or small molecules which bind to the protein so thattranscriptional activation mediated by the protein is prevented. Theseagents can be selected and screened 1) at random, 2) by a rationalselection or 3) by design using for example, protein or ligand modelingtechniques (preferably, computer modeling).

For random screening, agents such as antibodies, peptides,carbohydrates, pharmaceutical agents and the like are selected at randomand are assayed for their ability to bind to or stimulate/block theactivity of the androgen receptor splice variants.

Alternatively, agents may be rationally selected or designed. As usedherein, an agent is said to be “rationally selected or designed” whenthe agent is chosen based on the configuration of the androgen receptorsplice variant protein.

In one aspect, the invention provides a method of screening for an agentwhich modulates the activity of an androgen receptor splice variant,e.g., an agonist or antagonist, comprising: (a) contacting the androgenreceptor splice variant with the agent to be tested; and (b) assayingthe agent's effect on the androgen receptor splice variant activity. Insome embodiments, the activity to be tested is DNA binding, coactivatorbinding, binding to other transcription factors, and/or transcriptionalactivation.

In some embodiments, the agonist or antagonist is assayed to determinewhether DNA binding of the androgen receptor splice variants to anandrogen receptor response element is affected. In some embodiments,compounds that inhibit DNA binding would function as antagonists andcompounds that enhance DNA binding would function as agonists. Suchassays can be conducted in vitro, e.g., by electrophoretic mobilityshift assays (EMSAs) and can be employed to assay DNA binding of theandrogen receptor splice variants. In some embodiments, a method ofscreening for an antagonist or agonist which stimulates or blocks theDNA binding of the androgen receptor splice variant to DNA is provided,comprising: (a) contacting the androgen receptor splice variant with anagent to be tested and a nucleic acid sequence harboring a region thatbinds to the androgen receptor splice variant, such as an androgenreceptor response element; and (b) assaying binding of the androgenreceptor splice variant to the nucleic acid sequence. In someembodiments, auxiliary transcription factors, coactivator proteins,androgen receptor responsive promoter sequence optionally linked to atarget or reporter gene, nucleotides, and the like may be added.

In some embodiments, a method of screening for an antagonist or agonistwhich stimulates or blocks the binding of the androgen receptor splicevariant to one or more coactivator proteins is provided, comprising: (a)contacting the androgen receptor splice variant with an agent to betested and a coactivator protein; and (b) assaying binding of theandrogen receptor splice variant to the coactivator protein. Auxiliarytranscription factors, coactivators, androgen receptor responsivepromoter sequence optionally linked to a target or reporter gene,nucleotides, and the like may be added. Such binding of the androgenreceptor splice variants and coactivators can also be verified by EMSAs,provided, however, nucleic acid capable of binding either thecoactivator or androgen receptor splice variant is also added.

In some embodiments, the present invention relates to a method ofscreening for an antagonist or agonist which modulates the activity ofthe androgen receptor splice variant comprising: (a) contacting a cellexpressing the androgen receptor splice variant with an agent to betested; and (b) assaying expression of a gene mediated by the androgenreceptor splice variant. In some embodiments, mRNA levels (or cDNA) isassayed. In other embodiments, protein levels are assayed.

Any cell may be used in the above assay so long as it expresses afunctional form of an androgen receptor splice variant and the androgenreceptor splice variant activity can be measured. The preferredexpression cells are eukaryotic cells or organisms. Such cells can bemodified to contain DNA sequences encoding androgen receptor splicevariant using routine procedures known in the art. Alternatively, oneskilled in the art can introduce mRNA encoding the androgen receptorsplice variant protein directly into the cell.

In some embodiments, the assay is carried out in a cell-free system. Insome embodiments, a method of screening for an antagonist or agonistwhich modulates the activity of the androgen receptor splice variant isprovided, comprising: (a) contacting a cell-free system comprising theandrogen receptor splice variant with an agent to be tested; and (b)assaying expression of a gene mediated by the androgen receptor splicevariant. In some embodiments, mRNA levels (or cDNA) is assayed. In otherembodiments, protein levels are assayed. Auxiliary transcriptionfactors, coactivator complexes, androgen receptor responsive promoterslinked to a target or reporter gene, nucleotides, and the like may beadded.

Using androgen receptor splice variant ligands (ligands includingantagonists and agonists as described above) the present inventionfurther provides a method for modulating the activity of the androgenreceptor splice variant protein in a cell. In general, ligands(antagonists and agonists) which have been identified to block orstimulate the activity of androgen receptor splice variant can beformulated so that the ligand can be contacted with a cell expressing anandrogen receptor splice variant protein in vivo. The contacting of sucha cell with such a ligand results in the in vivo modulation of theactivity of the androgen receptor splice variant proteins. So long as aformulation barrier or toxicity barrier does not exist, ligandsidentified in the assays described above will be effective for in vivouse.

In another embodiment, the present invention relates to a method ofadministering androgen receptor splice variant or an androgen receptorsplice variant (including androgen receptor splice variant antagonistsand agonists) to an animal (preferably, a mammal (specifically, ahuman)) in an amount sufficient to effect an altered level of androgenreceptor splice variant in the animal. The administered androgenreceptor splice variant or androgen receptor splice variant ligand couldspecifically effect androgen receptor splice variant associatedfunctions. Further, since androgen receptor splice variant protein isexpressed in prostatic cancer cells, administration of androgen receptorsplice variant or androgen receptor splice variant ligand could be usedto alter androgen receptor splice variant levels in such cells.

In addition to screening for agonists and antagonists of the protein,the present invention also encompasses a screening system for detectingcompounds which bind to the DNA identified by SEQ ID NOS:1, 2, 3, 4, 35,36, or 38 encoding the androgen receptor splice variants, therebyinhibiting expression of the androgen receptor. In some embodiments, theDNA encoding the androgen receptor is coupled to a reporter system andis a marker for compounds exhibiting regulating properties of expressionof the messenger RNA.

One skilled in the art will appreciate that the amounts to beadministered for any particular treatment protocol can readily bedetermined. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of disease in the patient, counter indications, if any, andother such variables, to be adjusted by the individual physician. Dosagecan vary from 0.001 mg/kg to 50 mg/kg of androgen receptor splicevariant or androgen receptor splice variant ligand, in one or moreadministrations daily, for one or several days. Androgen receptor splicevariant or androgen receptor splice variant ligand can be administeredparenterally by injection or by gradual perfusion over time. It can beadministered intravenously, intraperitoneally, intramuscularly, orsubcutaneously.

Preparations for parenteral administration include sterile or aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose and sodium chloride, lactated Ringer's, or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives can also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like. See, generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds. (1980).

VIII. Transgenic Androgen Receptor Splice Variant Non-Human Animals

Methods of Generating Transgenic Non-Human Animals

The non-human animals of the invention comprise any animal having atransgenic interruption or alteration of the endogenous gene(s)(knock-out animals) and/or into the genome of which has been introducedone or more transgenes that direct the expression of human androgenreceptor splice variant protein. Also preferred are the introduction ofantisense androgen receptor splice variant nucleic acids.

Such non-human animals include vertebrates such as rodents, non-humanprimates, sheep, dog, cow, amphibians, reptiles, etc. Preferrednon-human animals are selected from non-human mammalian species ofanimals, most preferably, animals from the rodent family including ratsand mice, most preferably mice.

The transgenic animals of the invention are animals into which has beenintroduced by nonnatural means (i.e., by human manipulation), one ormore genes that do not occur naturally in the animal, e.g., foreigngenes, genetically engineered endogenous genes, etc. The nonnaturallyintroduced genes, known as transgenes, may be from the same or adifferent species as the animal but not naturally found in the animal inthe configuration and/or at the chromosomal locus conferred by thetransgene. Transgenes may comprise foreign DNA sequences, i.e.,sequences not normally found in the genome of the host animal.Alternatively or additionally, transgenes may comprise endogenous DNAsequences that are abnormal in that they have been rearranged or mutatedin vitro in order to alter the normal in vivo pattern of expression ofthe gene, or to alter or eliminate the biological activity of anendogenous gene product encoded by the gene. (Watson, J. D., et al., inRecombinant DNA, 2d Ed., W.H. Freeman & Co., New York (1992), pages 255272; Gordon, J. W., Intl. Rev. Cytol. 115:171 229 (1989); Jaenisch, R.,Science 240:1468 1474 (1989); Rossant, J., Neuron 2:323 334 (1990)).

In some embodiments, the transgenic non-human animals of the inventionare produced by introducing transgenes into the germline of thenon-human animal. Embryonic target cells at various developmental stagesare used to introduce the transgenes of the invention. Different methodsare used depending on the stage of development of the embryonic targetcell(s).

In some embodiments, the transgenic animal is made using the modifiedprobasin promoter ARR2PB which is positively regulated by androgen andexpressed in the prostate epithelium of sexually mature mice. In someembodiments, the invention is directed to a transgenic animal or itsprogeny or part thereof, which comprises in its genome a transgeneconstruct comprising a polynucleotide encoding an androgen receptorsplice variant encoding a polypeptide of the invention, such as apolypeptide selected from the group consisting of SEQ ID NO: 5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:37. In some embodiments,the transgenic animal is a mouse that expresses AR3. In someembodiments, the transgenic mouse is AR3Tg described below in Example13.

Transgenes may be introduced into non-human animals in order to provideanimal models for human diseases. Transgenic animals harboring one ormore of the androgen receptor splice variants of the invention areparticularly useful as an animal models for prostate cancer,particularly, androgen refractory prostate cancer. In some embodiments,the transgenic animal is transgenic for AR3, 4, 5 and 8. In otherembodiments, the transgenic animal is transgenic for AR3, 4 and 5. Inother embodiments, the transgenic animal is transgenic for AR3, 4, and8. In other embodiments, the transgenic animal is transgenic for AR3, 5and 8. In other embodiments, the transgenic animal is transgenic for AR3and 4. In other embodiments, the transgenic animal is transgenic for AR3and 5. In other embodiments, the transgenic animal is transgenic for AR3and 8. In other embodiments, the transgenic animal is transgenic forAR4, 5 and 8. In other embodiments, the transgenic animal is transgenicfor AR4 and 8. In other embodiments, the transgenic animal is transgenicfor AR4 and 5. In other embodiments, the transgenic animal is transgenicfor AR5 and 8. Animals that are transgenic for AR4b can also be made,alone, or in combination with the other AR splice variants shown above.

IX. Methods of Treatment

In some embodiments, these novel androgen receptor splice variantsherein described can be altered by methods and compositions. Forexample, gene delivery and gene silencing methods may be used to enhanceor inhibit the transcription or translation of these androgen receptorsplice variants AR3, AR4, AR4b, AR5 and AR8. Expression of thesedifferent androgen receptor splice variants may also be altered byincreasing or decreasing the stability of mRNA coding for AR3, AR4,AR4b, AR5 and AR8. The activity of these androgen receptor splicevariants may be altered by “gene silencing methods” which are generallyregarded as methods that prevent or decrease the rate of transcriptionor translation of a protein within a cell. Such gene silencing methodsinclude, but are not limited to antisense technology, RNA inhibitiontechnology (RNAi) and inactivation or degradation of transcriptionfactors required for androgen receptor (AR3, AR4, AR4b, AR5 or AR8)transcription, etc.

In some embodiments, the invention is directed to methods of treating orpreventing androgen refractory prostate cancer in a patient, comprisingadministering to the patient in need thereof an effective amount of anagent that inhibits the function of an androgen receptor splice variantselected from the group consisting of AR3, AR4, AR4b, AR5 and AR8. Insome embodiments, the agent inhibits expression of the androgen receptorsplice variant mRNA, and is selected from the group consisting ofantisense oligonucleotide, RNA interference oligonucleotide (RNAi),ribozyme or an agent that degrades transcription factors required forandrogen receptor transcription. In some embodiments, the agent is anantagonist that inhibits androgen-independent transcriptional activationby the androgen receptor splice variant. In some embodiments, the agentis an antibody that binds to the androgen receptor splice variant.

In some embodiments, the invention is directed to methods for inhibitingthe function of an androgen receptor splice variant in a prostate cancercell, comprising delivering an agent to the cell selected from the groupconsisting of an inhibitor of transcription of the androgen receptorsplice variant, an inhibitor of translation of the androgen receptorsplice variant, and an antagonist of the androgen receptor splicevariant, wherein the androgen receptor splice variant is selected fromthe group consisting of AR3, AR4, AR4b, AR5 and AR8.

Antisense oligonucleotides have been described as naturally occurringbiological inhibitors of gene expression in both prokaryotes (Mizuno etal., Proc. Natl. Acad. Sci. USA 81:1966-1970 (1984)) and eukaryotes(Heywood, Nucleic Acids Res. 14:6771-6772 (1986)), and these sequencespresumably function by hybridizing to complementary mRNA sequences,resulting in hybridization arrest of translation (Paterson, et al.,Proc. Natl. Acad. Sci. USA, 74:4370-4374 (1987)).

Thus, another gene therapy approach utilizes antisense technology.Antisense oligonucleotides are short synthetic DNA or RNA nucleotidemolecules formulated to be complementary to a specific gene or RNAmessage. Through the binding of these oligomers to a target DNA or mRNAsequence, transcription or translation of the gene can be selectivelyblocked and the disease process generated by that gene can be halted(see, for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press (1989)). The cytoplasmiclocation of mRNA provides a target considered to be readily accessibleto antisense oligodeoxynucleotides entering the cell; hence much of thework in the field has focused on RNA as a target. Currently, the use ofantisense oligodeoxynucleotides provides a useful tool for exploringregulation of gene expression in vitro and in tissue culture(Rothenberg, et al., J. Natl. Cancer Inst. 81:1539-1544 (1989)).

Antisense therapy is the administration of exogenous oligonucleotideswhich bind to a target polynucleotide located within the cells. Forexample, antisense oligonucleotides may be administered systemically foranticancer therapy (Smith, International Application Publication No. WO90/09180).

The antisense oligonucleotides of the present invention includederivatives such as S-oligonucleotides (phosphorothioate derivatives orS-oligos, see, Jack Cohen, supra). S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention may beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide which is a sulfur transferreagent. See Iyer et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer etal., J. Am. Chem. Soc. 112:1253-1254 (1990), the disclosures of whichare fully incorporated by reference herein.

The antisense oligonucleotides of the present invention may be RNA orDNA that is complementary to sequences within SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:38 andstably hybridize with such sequences that are specific for androgenreceptor splice variants of the invention. Use of an oligonucleotidecomplementary to such regions allows for selective hybridization toandrogen receptor splice variant mRNA.

In some embodiments, the antisense oligonucleotides of the presentinvention are a 15 to 30-mer fragment of the antisense DNA moleculecoding for sequences of the androgen receptor splice variant cDNAs.Preferred antisense oligonucleotides bind to the 5′-end of the androgenreceptor splice variant mRNAs. Such antisense oligonucleotides maybeused to down regulate or inhibit expression of the gene.

Other criteria that are known in the art may be used to select theantisense oligonucleotides, varying the length or the annealing positionin the targeted sequence.

Included as well in the present invention are pharmaceuticalcompositions comprising an effective amount of at least one of theantisense oligonucleotides of the invention in combination with apharmaceutically acceptable carrier. In one embodiment, a singleantisense oligonucleotide is utilized.

In another embodiment, two antisense oligonucleotides are utilized whichare complementary to adjacent regions of the genome. Administration oftwo antisense oligonucleotides that are complementary to adjacentregions of the genome or corresponding mRNA may allow for more efficientinhibition of genomic transcription or mRNA translation, resulting inmore effective inhibition of protein or mRNA production.

Preferably, the antisense oligonucleotide is coadministered with anagent which enhances the uptake of the antisense molecule by the cells.For example, the antisense oligonucleotide may be combined with alipophilic cationic compound which may be in the form of liposomes. Theuse of liposomes to introduce nucleotides into cells is taught, forexample, in U.S. Pat. Nos. 4,897,355 and 4,394,448, the disclosures ofwhich are incorporated by reference in their entirety (see also U.S.Pat. Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567,4,247,411, and 4,814,270 for general methods of preparing liposomescomprising biological materials).

Alternatively, the antisense oligonucleotide may be combined with alipophilic carrier such as any one of a number of sterols includingcholesterol, cholate and deoxycholic acid. A preferred sterol ischolesterol.

In addition, the antisense oligonucleotide maybe conjugated to a peptidethat is ingested by cells. Examples of useful peptides include peptidehormones, antigens or antibodies, and peptide toxins. By choosing apeptide that is selectively taken up by the targeted tissue or cells,specific delivery of the antisense agent maybe effected. The antisenseoligonucleotide maybe covalently bound via the 5′OH group by formationof an activated aminoalkyl derivative. The peptide of choice may then becovalently attached to the activated antisense oligonucleotide via anamino and sulfhydryl reactive hetero bifunctional reagent. The latter isbound to a cysteine residue present in the peptide. Upon exposure ofcells to the antisense oligonucleotide bound to the peptide, thepeptidyl antisense agent is endocytosed and the antisenseoligonucleotide binds to the target mRNA to inhibit translation(Haralambid et al., WO 8903849 and Lebleu et al., EP 0263740).

The antisense oligonucleotides and the pharmaceutical compositions ofthe present invention may be administered by any means that achievetheir intended purpose. For example, administration may be byparenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,or transdermal routes. The dosage administered will be dependent uponthe age, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired.

Compositions within the scope of this invention include all compositionswherein the antisense oligonucleotide is contained in an amounteffective to achieve the desired effect, for example, inhibition ofproliferation and/or stimulation of differentiation of the subjectcancer cells.

Alternatively, antisense oligonucleotides can be prepared which aredesigned to interfere with transcription of the gene by bindingtranscribed regions of duplex DNA (including introns, exons, or both)and forming triple helices (e.g., see Froehler et al., WO 91/06626 orToole, WO 92/10590). Preferred oligonucleotides for triple helixformation are oligonucleotides which have inverted polarities for atleast two regions of the oligonucleotide (Id.). Such oligonucleotidescomprise tandem sequences of opposite polarity such as 3′ - - -5′-L-5′ - - - 3′, or 5′ - - - 3′-L-3′ - - - 5′, wherein L represents a0-10 base oligonucleotide linkage between oligonucleotides. The invertedpolarity form stabilizes single-stranded oligonucleotides to exonucleasedegradation (Froehler et al., supra). The criteria for selecting suchinverted polarity oligonucleotides is known in the art, and suchpreferred triple helix-forming oligonucleotides of the invention arebased upon SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:35, SEQ ID NO:36 or SEQ ID NO:38.

In therapeutic application, the triple helix-forming oligonucleotidescan be formulated in pharmaceutical preparations for a variety of modesof administration, including systemic or localized administration, asdescribed above.

The antisense oligonucleotides of the present invention may be preparedaccording to any of the methods that are well known to those of ordinaryskill in the art, as described above.

Generally, RNAi technology is limited to tissue-specific ororgan-specific areas of the subject using tissue specific gene promotersor transcription factors. Promoters are nucleic acids that are generallylocated in the 5′ region of a gene, proximal to the start codon ornucleic acid which encodes untranslated RNA. The transcription of anadjacent nucleic acid segment is initiated at the promoter region. Anysuitable promoter may be used to control the production of RNA from thenucleic acid molecules of the invention. Promoters may be thoserecognized by any polymerase enzyme; for example, promoters may bepromoters for RNA polymerase II or polymerase III. Suitable promotersare known in the art and are within the scope of the present invention.Recombinant DNA methods, such as those that might be used to prepareconstructs of a tissue-specific promoter operably linked to a codingregion coding for mRNA are well known in the art.

The dsRNA molecules are digested in vivo to 21-23 nt fragment smallinterfering RNAs (siRNAs) which mediate the RNAi effect. In C. elegansand Drosophila, RNAi is induced by delivery of long dsRNA (up to 1-2 kb)produced by in vitro transcription. In mammalian cells, introduction oflong dsRNA elicits a strong antiviral response that blocks anygene-specific silencing. However, introduction of 21 nt siRNAs with 2 nt3′ overhangs into mammalian cells does not stimulate the antiviralresponse and effectively targets specific mRNAs for gene silencing. Thespecificity of this gene silencing mechanism is extremely high, blockingexpression only of targeted genes, while leaving other genes unaffected.Expression of androgen receptor splice variant transcripts of theinvention may be turned off, for example, by delivery of siRNAs orvectors encoding the same into gonads or early embryos. In anotherembodiment, the siRNAs are delivered to cells or tissues to turn offexpression of one or more androgen receptor splice variants. In oneembodiment, the cells are androgen refractory prostate cancer cells. Theartisan will appreciate that the siRNAs may be delivered to cells usingan in vivo or ex vivo approach. Preferred ex vivo approaches involvetransferring siRNAs to blood cells, bone marrow-derived cells, or stemcells.

The siRNAs or vectors encoding the same may be delivered to cells bytechniques known in the art as described above. Further, the siRNAs maybe prepared by any methods that are known in the art, including, but notlimited to, oligonucleotide synthesis, in vitro transcription,ribonuclease digestion, or generation of siRNAs in vivo. In oneembodiment, the siRNAs may be produced from vectors that are introducedinto cells. The vectors may be introduced by any known methods in theart, including but not limited to transfection, electroporation, orviral delivery systems. In some embodiments, the vectors are thepSilencer siRNA expression vectors, pSilencer 2.0-U6 and pSilencer3.0-H1. In a further embodiment, transcription of the siRNAs is drivenby a RNA polymerase III (pol III) promoter. The pol III promoter may bederived from any gene that is under the control of RNA polymerase III,including but not limited to H1 or U6.

A small hairpin RNA or short hairpin RNA (shRNA), or nucleic acidsencoding them can also be introduced into cells to produce siRNA. Shorthairpin RNA is a sequence of RNA that makes a tight hairpin turn thatcan be used to silence gene expression via RNA interference. The shRNAhairpin structure is cleaved by the cellular machinery into siRNA, whichis then bound to the RNA-induced silencing complex (RISC). This complexbinds to and cleaves mRNAs which match the siRNA that is bound to it.

In some embodiments, the siRNAs of the invention are encoded bynucleotide sequences within SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:38. In one embodiment,the siRNAs are about 20-1000 nucleotides in length. In anotherembodiment, the siRNAs are about 20-500 nucleotides in length. Inanother embodiment, the siRNAs are about 20-100 nucleotides in length.In another embodiment, the siRNAs are about 20-50 nucleotides in length.In another embodiment, the siRNAs are about 21-23 nucleotides in length.The siRNAs may be produced by PCR amplification of genomic DNA or cDNA,using primers derived from androgen receptor splice variant sequence,and cloned into expression vectors for siRNA production. In anotherembodiment, oligonucleotides that correspond to androgen receptor splicevariant sequence may be chemically synthesized and inserted intoexpression vectors for siRNA production. The siRNAs or vectors encodingthe same are introduced into cells to block expression of the androgenreceptor splice variant polypeptides. siRNA can also be produced bychemical synthesis of oligonucleotide of RNA of 21-23 nucleotides. Insome embodiments, the androgen receptor splice variants to be knockeddown using RNAi are selected from the group consisting of AR3, AR4,Ar4b, AR5 and AR8. In some embodiments, the androgen receptor to beknocked down is AR3. In some embodiments, short hairpin RNA is used toknock down AR3. In some embodiments, the short hairpin RNA is shAR3-1(made using SEQ ID NOS:13 and 14) or shAR3-2 (made using SEQ IDNOs:15-16). In some embodiments, siRNA directed against SEQ ID NO:30encoding AR8 is used to knock down AR8 in cells.

In one embodiment, the siRNAs are composed of nucleotides A, G, T, C, orU. Additionally, the siRNAs may be composed of unusual or modifiednucleotides including but not limited to inosinic acid, 1-methylinosinic acid, 1-methyl guanylic acid, NN-dimethyl guanylic acid,pseudouridylic acid, ribothymidylic acid, 5-hydroxymethylcytosine, and5-hydroxymethyluridine. RNA may be synthesized either in vivo or invitro and later introduced into cells. Endogenous RNA polymerase of thecell may mediate transcription in vivo, or cloned RNA polymerase can beused for transcription in vitro. For transcription from a transgene invivo or an expression construct, a regulatory region (e.g., promoter,enhancer, silencer, splice donor and acceptor, polyadenylation) maybeused to transcribe the RNA strand (or strands); the promoters may beknown inducible promoters that respond to infection, stress,temperature, wounding, or chemicals. Inhibition may be targeted byspecific transcription in an organ, tissue, or cell type; stimulation ofan environmental condition (e.g., infection, stress, temperature,chemical inducers); and/or engineering transcription at a developmentalstage or age. The RNA strands may or may not be polyadenylated; the RNAstrands may or may not be capable of being translated into a polypeptideby a cell's translational apparatus. RNA may be chemically orenzymatically synthesized by manual or automated reactions. The RNA maybe synthesized by a cellular RNA polymerase or a bacteriophage RNApolymerase (e.g., T3, T7, SP6). The use and production of an expressionconstruct are known in the art (see, for example, WO 97/32016; U.S. Pat.Nos. 5,593,874; 5,698,425; 5,712,135; 5,789,214; and 5,804,693; and thereferences cited therein). If synthesized chemically or by in vitroenzymatic synthesis, the RNA may be purified prior to introduction intothe cell. For example, RNA can be purified from a mixture by extractionwith a solvent or resin, precipitation, electrophoresis, chromatography,or a combination thereof. Alternatively, the RNA may be used with no ora minimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

RNA containing nucleotide sequence identical to a fragment of theandrogen receptor splice variant sequences are preferred for inhibition;however, RNA sequences with insertions, deletions, and point mutationsrelative to the androgen receptor splice variant sequences of theinvention can also be used for inhibition. Sequence identity mayoptimized by sequence comparison and alignment algorithms known in theart (see Gribskov and Devereux, Sequence Analysis Primer, StocktonPress, 1991, and references cited therein) and calculating the percentdifference between the nucleotide sequences by, for example, theSmith-Waterman algorithm as implemented in the BESTFIT software programusing default parameters (e.g., University of Wisconsin GeneticComputing Group). Alternatively, the duplex region of the RNA may bedefined functionally as a nucleotide sequence that is capable ofhybridizing with a fragment of the target gene transcript.

In some embodiments, the invention is directed to a method for treatingcancer in an animal comprising administering to the animal an isolatedshort interfering RNA molecule that inhibits expression of an androgenreceptor splice variant selected from the group consisting of AR3 (SEQID NO:1), AR4 (SEQ ID NO:2 or 35), AR4b (SEQ ID NO:36 or 38), AR5 (SEQID NO:3) and AR8 (SEQ ID NO:4).

Ribozymes may also be used for gene silencing. For example, antisenseRNA/ribozyme fusions which comprise (1) antisense RNA corresponding to atarget gene and (2) one or more ribozymes which cleave RNA can be used,as well as vectors which express these fusions, methods for producingthese vectors, and methods for using these vectors.

Preferred targets for ribozymes are androgen receptor splice variantnucleotide sequences. In some embodiments, the ribozyme molecule of thepresent invention is designed based upon the chloramphenicolacetyltransferase ribozyme or hairpin ribozymes. Alternatively, ribozymemolecules are designed as described by Eckstein et al., (InternationalPublication No. WO 92/07065) who disclose catalytically active ribozymeconstructions which have increased stability against chemical andenzymatic degradation, and thus are useful as therapeutic agents.

In an alternative approach, an external guide sequence (EGS) can beconstructed for directing the endogenous ribozyme, RNase P, tointracellular mRNA, which is subsequently cleaved by the cellularribozyme (Altman et al., U.S. Pat. No. 5,168,053). Preferably, the EGScomprises a ten to fifteen nucleotide sequence complementary to an mRNAand a 3′-NCCA nucleotide sequence, wherein N is preferably a purine(Id.). After EGS molecules are delivered to cells, as described below,the molecules bind to the targeted mRNA species by forming base pairsbetween the mRNA and the complementary EGS sequences, thus promotingcleavage of mRNA by RNase P at the nucleotide at the 5′ side of thebase-paired region (Id.).

Included as well in the present invention are pharmaceuticalcompositions comprising an effective amount of at least one ribozyme orEGS of the invention in combination with a pharmaceutically acceptablecarrier. Preferably, the ribozyme or EGS is coadministered with an agentwhich enhances the uptake of the ribozyme or EGS molecule by the cells.For example, the ribozyme or EGS may be combined with a lipophiliccationic compound which may be in the form of liposomes, as describedabove. Alternatively, the ribozyme or EGS may be combined with alipophilic carrier such as any one of a number of sterols includingcholesterol, cholate and deoxycholic acid. A preferred sterol ischolesterol.

The ribozyme or EGS, and the pharmaceutical compositions of the presentinvention may be administered by any means that achieve their intendedpurpose. For example, administration may be by parenteral, subcutaneous,intravenous, intramuscular, intra-peritoneal, or transdermal routes. Thedosage administered will be dependent upon the age, health, and weightof the recipient, kind of concurrent treatment, if any, frequency oftreatment, and the nature of the effect desired. For example, as much as700 milligrams of antisense oligodeoxynucleotide has been administeredintravenously to a patient over a course of 10 days (i.e., 0.05mg/kg/hour) without signs of toxicity (Sterling, “Systemic AntisenseTreatment Reported,” Genetic Engineering News 12(12):1, 28 (1992)).

Compositions within the scope of this invention include all compositionswherein the ribozyme or EGS is contained in an amount which is effectiveto achieve inhibition of proliferation and/or stimulate differentiationof the subject cancer cells, or alleviate AD. While individual needsvary, determination of optimal ranges of effective amounts of eachcomponent is with the skill of the art.

In addition to administering the antisense oligonucleotides, ribozymes,or EGS as a raw chemical in solution, the therapeutic molecules may beadministered as part of a pharmaceutical preparation containing suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the antisenseoligonucleotide, ribozyme, or EGS into preparations which can be usedpharmaceutically.

Suitable formulations for parenteral administration include aqueoussolutions of the antisense oligonucleotides, dsRNAs, ribozymes, EGS inwater-soluble form, for example, water-soluble salts. In addition,suspensions of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, ethyl oleate or triglycerides. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

Alternatively, antisense RNA molecules, ribozymes, and EGS can be codedby DNA constructs which are administered in the form of virions, whichare preferably incapable of replicating in vivo (see, for example,Taylor, WO 92/06693). For example, such DNA constructs may beadministered using herpes-based viruses (Gage et al., U.S. Pat. No.5,082,670). Alternatively, antisense RNA sequences, ribozymes, and EGScan be coded by RNA constructs which are administered in the form ofvirions, such as retroviruses. The preparation of retroviral vectors iswell known in the art (see, for example, Brown et al., “RetroviralVectors,” in DNA Cloning: A Practical Approach, Volume 3, IRL Press,Washington, D.C. (1987)).

Specificity for gene expression may be conferred by using appropriatecell-specific regulatory sequences, such as cell-specific enhancers andpromoters. Such regulatory elements are known in the art, and their useenables therapies designed to target specific tissues, such as liver,lung, prostate, kidney, pancreas, etc., or cell populations, such aslymphocytes, neurons, mesenchymal, epithelial, muscle, etc.

In the method of treating an androgen receptor splice variant-associateddisease (preferably, prostate cancer, in particular, androgen refractoryprostate cancer) in a patient in need of such treatment, in someembodiments, gene replacement (“knock out”) technology is used thatwould replace or delete the disease causing androgen receptor splicevariant sequences to treat the disease (specifically, prostate cancer orandrogen refractory prostate cancer).

Included as well in the invention are pharmaceutical compositionscomprising an effective amount of at least one androgen receptor splicevariant antisense oligonucleotide, in combination with apharmaceutically acceptable carrier. Such antisense oligos include, butare not limited to, at least one nucleotide sequence which iscomplementary to exons encoding androgen receptor splice variants; a DNAsequence of SEQ ID NO:1, 2, 3, 4, 35, 36 or 38; or a DNA sequenceencoding at least 4 amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:38.

Alternatively, the androgen receptor splice variant nucleic acid can becombined with a lipophilic carrier such as any one of a number ofsterols including cholesterol, cholate and deoxycholic acid. A preferredsterol is cholesterol.

The androgen receptor splice variant nucleic acids and thepharmaceutical compositions of the invention can be administered by anymeans that achieve their intended purpose. For example, administrationcan be by parenteral, subcutaneous, intravenous, intramuscular,intra-peritoneal, or transdermal routes. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

Compositions within the scope of this invention include all compositionswherein the androgen receptor splice variant antisense oligonucleotideis contained in an amount effective to achieve decreased expression ofthe androgen receptor splice variant mRNA encoded by the androgenreceptor gene. While individual needs vary, determination of optimalranges of effective amounts of each component is within the skill of theart. Typically, the androgen receptor splice variant nucleic acid can beadministered to mammals, e.g. humans, at a dose of 0.005 to 1 mg/kg/day,or an equivalent amount of the pharmaceutically acceptable salt thereof,per day of the body weight of the mammal being treated.

Suitable formulations for parenteral administration include aqueoussolutions of the androgen receptor splice variant nucleic acid inwater-soluble form, for example, water-soluble salts. In addition,suspensions of the active compounds as appropriate oily injectionsuspensions can be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, ethyl oleate or triglycerides. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension can alsocontain stabilizers.

Many vector systems are known in the art to provide such delivery tohuman patients. For example, retrovirus systems can be used, especiallymodified retrovirus systems and especially herpes simplex virus systems(Gage et al., U.S. Pat. No. 5,082,670). Such methods are provided for,in, for example, the teachings of Breakefield, X. A. et al., The NewBiologist 3:203 218 (1991); Huang, Q. et al., Experimental Neurology115:303 316 (1992); WO93/03743; WO90/0944; Taylor, WO 92/06693;Mulligan, R. C., Science 260:926 932 (1993); and Brown et al.,“Retroviral Vectors,” in DNA Cloning: A Practical Approach, Volume 3,IRL Press, Washington, D.C. (1987).

The means by which the vector carrying the nucleic acid can beintroduced into the cell include but is not limited to, microinjection,electroporation, transduction, or transfection using DEAE-Dextran,lipofection, calcium phosphate or other procedures known to one skilledin the art (Molecular Cloning, A Laboratory Manual, Sambrook et al.,eds., Cold Spring Harbor Press, Plainview, N.Y. (1989)).

Further methods which can be used to transfer nucleic acid to a patientare set forth in Chatterjee and Wong, Current Topics in Microbiol. andImmuno., 218: 61 73 (1996); Zhang, J. Mol. Med. 74:191 204 (1996);Schmidt-Wolf and Schmidt-Wolf, J. of Hematotherapy 4:551 561 (1995);Shaughnessy et al., Seminars in Oncology 23 (1): 159 171 (1996); andDunbar Annu. Rev. Med. 47:11 20 (1996).

Specificity for gene expression in prostate cancer cells can beconferred by using appropriate cell-specific regulatory sequences, suchas cell-specific enhancers and promoters.

Other methods of inhibiting the activity of these novel androgenreceptor splice variants include the use of antibodies or functionalfragments, or other antagonists which bind the androgen receptor. Asused herein, the term antibody includes at least monoclonal antibodiesand polyclonal antibodies and functional fragments of an antibody.Antibodies or functional fragments thereof can be used as antagonists ofAR3, AR4, AR4b, AR5, or AR8. Use of functional fragments, such as theFab, Fab′, or F(ab′)₂ fragments are often suitable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

Antibodies are prepared by well-known methods in the art, such asimmunizing suitable mammalian hosts in appropriate immunizationprotocols using AR fragments or peptides or polypeptides. Thesepeptides, polypeptides or fragments can be conjugated to suitablecarriers such as BSA (bovine serum albumin), KLH (Keyhole limpethemocyanin), or the like.

While the polyclonal antisera produced in this way be satisfactory forsome applications, for pharmaceutical application, use of monoclonalpreparations is also suitable. Immortalized cell lines which secrete thedesired monoclonal antibodies may be prepared using the standard methodof Kohier and Milstein or modification which effect immortalization oflymphocytes or spleen cells, as is generally known. The immortalizedcell lines secreting the desired antibodies are screened by immunoassayin which the antigen is the peptide hapten, polypeptide or protein. Whenthe appropriate immortalized cell culture secreting the desired antibodyis identified, the cells can be cultured in vitro or by production inascites fluid. The desired monoclonal antibodies can then be recoveredfrom the culture supernatant.

The antibodies or fragments may also be produced by recombinant methods.Regions that bind specifically to the desired regions of the AR or itsdownstream effector molecules can also be produced in the context ofchimeras with multiple species origin. Humanized and fully humanantibodies, such as those identified by phage display or produced by aXenomouse, are also contemplated. Antibody reagents so created arecontemplated for use diagnostically or as stimulants or inhibitors ofthe activity of androgen receptor (AR) splice variants herein described.

In some embodiments, the antibodies are directed against AR3 (SEQ IDNO:5), AR4 (SEQ ID NO:6), AR4b (SEQ ID NO: 37), AR5 (SEQ ID NO:7) or AR8(SEQ ID NO:8) polypeptides.

Treatment comprises parenterally administering a single or multipledoses of the antibody, fragment or derivative. Preferred for humanpharmaceutical use are high affinity potent androgen receptor splicevariant-inhibiting and/or neutralizing murine and chimeric antibodies,fragments and regions of this invention.

In some embodiments, the invention is directed to methods for inhibitingthe activity of androgen splice receptor variants AR3, AR4, AR4b, AR5 orAR8 in a prostate cancer cell comprising administering to said cell anantibody or functional fragment of said antibody which binds theandrogen receptor.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adaily dosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10milligrams per kilogram per day given in divided doses 1 to 6 times aday or in sustained release form is effective to obtain desired results.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association witha pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes and nonaqueous vehicles such as fixedoils may also be used. The vehicle or lyophilized powder may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

The murine and chimeric antibodies, fragments and regions of thisinvention, their fragments, and derivatives can be used therapeuticallyas immunoconjugates (see for review: Dillman, R. O., Ann. Int. Med.111:592 603 (1989)). They can be coupled to cytotoxic proteins,including, but not limited to Ricin-A, Pseudomonas toxin, and Diphtheriatoxin. Toxins conjugated to antibodies or other ligands, are known inthe art (see, for example, Olsnes, S. et al., Immunol. Today 10:291 295(1989)). Plant and bacterial toxins typically kill cells by disruptingthe protein synthetic machinery.

The antibodies of this invention can be conjugated to additional typesof therapeutic moieties including, but not limited to, radionuclides,cytotoxic agents and drugs. Examples of radionuclides which can becoupled to antibodies and delivered in vivo to sites of antigen include²¹²Bi, ¹³¹I, ¹⁸⁶Re, and ⁹⁰Y, which list is not intended to beexhaustive. The radionuclides exert their cytotoxic effect by locallyirradiating the cells, leading to various intracellular lesions, as isknown in the art of radiotherapy.

Cytotoxic drugs which can be conjugated to antibodies and subsequentlyused for in vivo therapy include, but are not limited to, daunorubicin,doxorubicin, methotrexate, and Mitomycin C. Cytotoxic drugs interferewith critical cellular processes including DNA, RNA, and proteinsynthesis. For a fuller exposition of these classes of drugs which areknown in the art, and their mechanisms of action, see Goodman, A. G., etal., Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7thEd., Macmillan Publishing Co., 1985.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or murine and chimeric antibodies,fragments and regions, or with lymphokines or hematopoietic growthfactors, etc., which serve to increase the number or activity ofeffector cells which interact with the antibodies.

Application of the teachings of the present invention to a specificproblem or environment is within the capabilities of one having ordinaryskill in the art in light of the teaching contained herein. Examples ofthe products and processes of the invention appear in the followingnon-limiting Examples.

EXAMPLES Example 1 Identification of Novel Androgen-Independent ARAlternative Splicing Isoforms

We examined the expression of AR protein in a panel of PCA cell linesusing an antibody recognizing the N-terminus of AR. In addition to thewell characterized 110 kD AR protein, we detected one band approximately80 kD in the LNCaP derivative C-81, CWR-R1 and 22Rv1 cells which areknown to grow in the androgen-depleted medium (FIG. 1 a). This shortform of AR (ARs) appeared to correspond to the truncated AR previouslyreported in CWR-R1 and 22Rv1 cells. On the other hand, the 80 kD ARs wasbarely detectable in the androgen-dependent LAPC4 and LNCaP cells. Thesedata implied an inverse correlation between the ARs andandrogen-dependency of these cell lines in cell culture. To confirm thatthe 80 kD ARs was indeed derived from the AR gene, we treated CWR-R1with a panel of shRNAs specifically targeting distinct regions of the ARgene. These shRNAs could differentially knock down AR and ARs,suggesting that AR and ARs may be translated from more than onetranscript. ARs was efficiently knocked down by shARc targeting at the1st Exon but barely affected by the shARb or shARa targeting at Exon 5and 8 respectively. This finding implies that the transcript encodingthe ARs may contain Exon 1 but may not have intact Exons 5 and 8.Similar effects were also observed in the 22Rv1 cells (data not shown).These findings prompted us to clone possible alternative splice variantsof AR in these cells by using the 3′ RACE with a primer corresponding tothe shARc target sequence. As shown in FIG. 1 c, multiple PCR productsresulted from the 3′ RACE were detected. Subsequent cloning andsequencing analysis revealed that the major band around 2 kb turned outto be the 110 kD prototype AR. The other bands were found to be-theresult of alternative splicing through various mechanisms including exonskipping, cryptic splicing donor or acceptor usage, cryptic exoninclusion, etc. More than 20 splicing variants have been identified sofar. Among them, three variants (designated as AR3, AR4 and AR5) werepredicted to be translated into a protein around 80 kD, similar to theone we and other detected in CWR-R1 and 22Rv1 cells (FIGS. 11-13). Theschematic gene structures of these AR variants are shown in FIG. 1 d.They contain-the intact NTD and the DBD but lack the hinge region andthe LBD. Instead, they contain 16-53 unique amino acids, which are notpresent in AR, at their C-termini respectively. We then designed theisoform specific primers that only recognize the unique junctionsequence present in each isoform. These isoforms were detected in apanel of human benign and malignant prostate tissues by RT-PCR (FIG. 7).FIG. 1 e shows that AR3 appeared to be one of the most frequently andabundantly expressed iso forms detected in all three hormone refractorycell lines in our real-time PCR analysis. Consistent with the WesternBlot data in FIG. 1 a, the expression level of AR3 is significantlyincreased in the high passage androgen-independent LNCaP derivativeC-81, compared with the parental androgen-sensitive LNCaP cells (FIG. 1e right panel). We also detected an increase of AR3 expression inhormone refractory CWR22 xenografts compared to the hormone naïvecounterparts (FIG. 8), suggesting a role of AR3 in androgenindependence. Previous studies have shown that the deletion of theligand binding domain of AR leads to constitutive activation of itstranscriptional activity. Therefore, these AR splice variants may beconstitutively active in their host cells and exert its transcriptionalactivity in a true androgen-independent manner. The AR isoforms weretransiently transfected into COS-1 cells and their effects on theARE-containing promoter reporter activity were tested. As expected, allthe three AR iso forms induced androgen independent activation of thereporter. AR3 appeared to be most active in the luciferase assaycompared to the other two splice variants (FIG. 1 f). We thereforefocused our functional study on AR3. In addition, the AR3 activity wasincreased in a dose-dependent manner, however, unlike the prototype ARwhose activity was dramatically stimulated by addition of DHT, thetranscriptional activity of AR3 is completely independent of androgen(FIG. 1 g). We also overexpressed AR3 in LNCaP cells and examinedwhether its activity could be modulated by AR, androgen oranti-androgen. FIG. 1 h shows that inhibition of AR either by thespecific shRNA or casodex did not affect AR3 activity regardless of DHTtreatment while the activity of the endogenous AR or the exogenouscodon-switched wild-type AR (ARcs, as described previously (Guo, Z. etal., Cancer Cell 10, 309-19 (2006)) was induced by DHT and blocked bycasodex as expected. Thus, AR3 activity is not controlled by DHT,casodex or the status of AR, suggesting that AR3 may be a true androgenindependent transcription factor.

To further characterize the endogenous AR3, we developed a polyclonalantibody which specifically recognizes the unique sequence at theC-terminus of AR3. FIG. 2 a shows that the anti-AR3 antibody onlydetected the overexpressed AR3 but not AR in COS-1 cells. Knock-down ofAR3 expression in 22Rv1 and CWR-R1 cells by treatment with the specificshRNAs for AR3 diminished the immunoreactivity of the anti-AR3 as wellas the anti-AR with the 80 kD ARs but it had little effect on theanti-AR reactivity with the 110 kD AR (FIG. 2 b). The anti-AR3 antibodycould efficiently and selectively immunoprecipitate the endogenous AR3but not AR (FIG. 2 c). Immunofluorescence staining revealed that AR3 waspresent in both nucleus and cytoplasm in CWR-R1 and 22Rv1 cells (FIG. 2d). Western Blot analysis showed AR3 is expressed in all testedAR-positive prostate cancer cell lines. It is noteworthy that the levelof AR3 in the androgen-independent LNCaP derivatives C81, C4-2 and C4-2Bwas significantly higher than that in the androgen-sensitive parentalline (FIG. 3 a). Immunohistochemistry analysis on human prostate tissuemicroarrays revealed a marked change in AR3 expression level and patternin malignant prostate tissues compared to the benign counterparts (FIG.3 b). In benign prostate tissues, the anti-AR3 antibody mainly stainedthe basal cells as well as the stroma cells but most of luminal cellswere barely stained (the mean cytoplasmic staining score 1.52±0.34)(FIG. 3 c and FIG. 10). On the other hand, the majority of luminal cellsin the malignant glands showed strong cytoplasmic staining by the AR3antibody (mean score 4.74±0.13). In addition, a significantredistribution of AR3 protein to the nucleus was observed in the hormonerefractory tumor tissue samples (44% nuclear positive) compared to thehormone naïve counterparts (9% nuclear positive). Thus, nucleartranslocation of AR3 is significantly increased in hormone refractoryprostate tumors. To assess whether AR3 could be used as a potentialprognostic marker for prostate cancer, clinical outcome analysis wasperformed on 224 PCA patient samples with clinicopathologicalinformation. Patients with elevated PSA levels following radicalprostatectomy are at a high risk to develop distant metastases and dieof prostate cancer. Clinical failure was defined as a PSA elevation ofgreater than 0.2 ng/ml following radical prostatectomy with successiveincreasing PSA values. Kaplan-Meier analysis indicated that prostatecancer patients that have higher cytoplasmic staining of AR3 (stainingscore>=6) have a greater risk for PSA recurrence after radicalprostatectomy (FIG. 3 d, log-rank test, p<0.0001). To test whetheroverexpression of AR3 in prostate cancer cells could conferandrogen-independent growth, LNCaP cells were infected with thelentivirus encoding AR3 and maintained in the medium supplemented withthe charcoal-stripped serum. As shown in FIG. 4 a, overexpression of AR3in LNCaP cell promoted growth in androgen-depleted medium. Such growthenhancement was also observed in the castrated SCID mice xenograftmodels (FIG. 4 b). We further investigated whether expression of AR3 inhormone refractory prostate cancer cells is required forandrogen-independent growth by specifically knocking down AR3. As shownin FIGS. 4 c and 4 d, treatment of both 22Rv1 and CWR-R1 cells with thelentivirus encoding the shRNA specific for AR3 attenuated their growthin the androgen-depleted medium as well as in the castrated nude mice,suggesting that AR3 activity is required for prostate cancer cell growthin both cell culture and xenograft models under androgen-depletedconditions. It should be noted that knock-down of AR3 in these cells didnot alter the expression of AR, therefore, AR3 may play an indispensablerole in promoting androgen-independent growth of prostate cancer,possibly through regulating a different subset of target genes that arenot shared with AR.

To identify potential AR3 target genes in hormone refractory prostatecancer cells, we selectively knocked down AR3 or AR by the specificshRNAs in two different cell lines CWR-R1 and 22Rv1. The differentialgene expression resulted from AR3 or AR knockdown were determined bymicroarray analysis. The differential expression of a set of 188 geneswas consistently detected in both cell lines when AR3 was specificallyknocked down while the expression of 412 genes was altered in both celllines when AR was specifically inhibited (FIG. 5 a). Among them, 71genes are commonly regulated by both AR and AR3. A partial list of thesegenes is summarized in Table 1. Several known AR target genes such asIGFBP3 and FKBP5 are also regulated by AR3. However, many classical ARtarget genes such as CLU, TMEPAI, KLK3 (PSA) and CLDN4 were not affectedby AR3 knock-down (Table 2). Among the 117 genes that are exclusivelyregulated by AR3 (Table 3), there are a number of genes, such as MAP4K4,Mdm4, TAF9B, HOXB7 and ELKJ, have been found to be upregulated inhormone refractory or metastatic prostate cancers in previous geneprofiling studies on human tissue or xenograft samples (Chen, C. D. etal., Nat Med 10, 33-9 (2004); Best, C. J. et al., Clin Cancer Res 11,6823-34 (2005); Varambally, S. et al., Cancer Cell 8, 393-406 (2005)).Interestingly, the serine/threonine kinase AKT1, which has beenimplicated in prostate cancer development and progression, appeared tobe regulated by AR3 as well. We further validated the microarray resultsby the real-time PCR. As shown in FIG. 5 b, the level of AKT1 transcriptin AR3-knockdown cells is significantly less than that in the cellstreated with the control scrambled shRNA or the shRNA specific for AR.The protein level of AKT1 was also reduced accordingly in AR3-knockdownCWR-R1 cells (FIG. 5 c). In addition, overexpression of AR3 in animmortalized human normal prostate epithelial cell line HPr-1, whichexpresses little or no AR3, led to an increase of AKT1 expression whileno effect was detected in HPr-1 cells overexpressing AR. We alsoexamined the level of AKT1 protein in the xenograft tumors described inFIG. 4. Consistent with the results in the cell lines, AKT1 proteinlevel is increased in the xenograft tumor of LNCaP overexpressing AR3and decreased in the xenograft tumor of 22Rv1 with AR3 knock-down (FIG.5 d), suggesting that the level of AKT1 may also be regulated by AR3 inthese xenograft tumors and correlated with the tumor growth underandrogen-depleted conditions. Furthermore, we identified at least twoputative ARE sites in the AKT1 regulatory region and showed that AR3 butnot AR was able to bind to these ARE sites determined by the chromatinimmunoprecipitation (ChIP) assays (FIG. 5 e), suggesting that AR3 maydirectly regulate AKT1 at the transcriptional level. On the other hand,AR3 failed to bind to the ARE site located at the regulatory region ofthe PSA gene, a well established AR target gene. Taken together, ourdata suggest that AR3 and AR may play an overlapping but yet distinctroles in prostate cells by regulating transcription of a subset ofcommon target genes as well as a different set of target genes unique toeach receptor. Aberrant expression of AR3 in prostate cancer maycontribute to androgen-independence in advanced PCA through regulationof a series of genes functioning in survival, dedifferentiation,chromatin remodeling and transcription specificity (FIG. 6).

Example 2 Cell Culture and Transfection

LNCaP, PC-3, 22Rv1 and COS-i cells were purchased from the American TypeCulture Collection. Hormone refractory prostate cancer cell lines C-S1,C4-2, C4-2B and CWR-R1 were kindly provided by Drs. Ming Fong Lin(Igawa, T. et al., Prostate 50, 222-35 (2002)), Donald Tindall,Christopher Gregory and Elizabeth Wilson (Gregory, C. W. et al., CancerRes 61, 2892-8 (2001))., respectively. LNCaP, PC-3, C-81, C4-2 and C4-2Bcells were maintained in RPMI 1640 with 10% FBS. COS-1 cells were grownin DMEM medium with 10% FBS. 22Rv1 and CWR-R1 cells were maintained inRPMI 1640 with 10% heat-inactivated FBS. LAPC-4 cells (kindly providedby Dr. Charles Swayers) were maintained in IMEM supplemented with 15%FBS and 10 nM DHT. The immortalized human normal prostate epithelialcell line HPr-1 described previously (Choo, C. K. et al., Prostate 40,150-8 (1999) was kindly provided by Dr. Patrick Ling and maintained inthe SEM keratinocyte medium.

The cells were transfected with FuGENE 6 (Roche) or LipofectAMINE 2000(Invitrogen) following the manufacturer's instruction.

Example 3 Antibodies

The antibodies used in immunoblot, immunoprecipitation, andimmunofluorescence are mouse monoclonal antiAkt1 (2H10) (CellSignaling), mouse monoclonal anti-AR (441), anti-actin (C2) and rabbitpolyclonal anti-AR (H-280) and anti-AR (C-19) (Santa Cruz). The anti-AR3antibody was developed by immunizing the rabbits with a syntheticpeptide corresponding to the C-terminal 16 unique amino acids of AR3 andthe terminal bleeds were affinity purified by a commercial carrier.

Example 4 Cloning and Constructs

The primer corresponding to the shARc target sequence (Guo109A,5′-CAGAGTCGCGACTACTACAACTTTCCA-3′) (SEQ ID NO:9) was used to amplify the3′ end of the AR transcripts in CWR-R1 and 22Rv1 cells using the5′/3′-Rapid Amplification of cDNA Ends (RACE) Kit (Roche AppliedScience) according to the manufacturer's instructions. PCRs were carriedout using Expand High Fidelity PCR system (Roche Applied Science) whichis composed of a unique enzyme mix containing thermostable Taq DNApolymerase and a proofreading polymerase. For the generation of PCRproducts with high fidelity and specificity, an initial 2-min.denaturation step at 94° C. was followed by 10 cycles of successiveincubations at 94° C. (15 s), 65° C. (30 s), and 68° C. (3 min) and 20cycles of successive incubations at 94° C. (15 s), 65° C. (30 s), and68° C. (initially 3 min with an increase of 5 s in each successivecycle), and a final 7-min elongation at 72° C. PCR products wereelectrophoresed on 1% agarose gels and the specific bands/DNA fragmentswere excised and purified with a Qiagen gel purification kit (Qiagene)according to the manufacturer's instruction. The purified fragments werecloned into the pCR2.1 vector using the PCR TA cloning kit (Invitrogen)and subsequently sequenced (Applied Biosystems sequencer Model 3730).The entire cDNA coding region of the AR isoforms was amplified from cDNAof CWR-R1 or 22Rv1 and first cloned into PCR2.1 vector using a TAcloning kit (Invitrogen). After 20 verification of the sequences, theresulting amplification products were subcloned into a lentivirusexpressing vector using BamHI site as described previously.³⁵

The shRNAs specific for human AR gene in the lentiviral pLKO. 1-purovector were purchased from Sigma. The sequences of these shRNAs wereprovided by the manufacture as following:

shARa: (SEQ ID NO: 10)CCGGCCTGCTAATCAAGTCACACATCTCGAGATGTGTGACTTGATTAGCA GGTTTTT; shARb: (SEQID NO: 11) CCGGCACCAATGTCAACTCCAGGATCTCGAGATCCTGGAGTTGACATTGG TGTTTT;

shARc: CCGGCGCGACTACTACAACTTTCCACTCGAGTGGAAAGTTGTAGTAGTCGCG UTTT (SEQ IDNO:12). The shRNAs specific for AR3 isoform (shAR3) were constructed asdescribed previously (Guo, Z. et al., Cancer Cell 10, 309-19 (2006)).The oligo sequences used were as follows: shAR3-1 (G622), Guo167a,TGTAATAGTGGTTACCACTCTTCAAGAGAGAGTGGTAACCACTATTACTTTTT TTTC-3′ (SEQ IDNO:13), Guo167b, TCGAGAAAAAAAAGTAATAGTGGTTACCACTCTCTCTTGAAGAGTGGTAACCACTATTACA-3′ (SEQ ID NO:14), shAR3-2 (G626), Guo171a,5′-TAGGCTAATGAGGTrTATTTCTCAAGAGAAATAAACCTCATTAGCCTTTTTTTT TC-3′ (SEQ IDNO: 15), Guo171b,5′-TCGAGAAAAAAAAAGGCTAATGAGGTTTATTTTCCTTGAGAAATAAACCTCA TTAGCCTA-3′ (SEQID NO:16); shAR3-sc (G627, AR3 scrambled control shRNA sequence):Guo172a, 5′-TAAGAAACAGTCCGACTCAATTCAAGAGATTGAGTCGGACTGTTTCTTTCTTTTTTC-3′ (SEQ ID NO:17); Guo172b, 5′-TCGAGAAAAAAGAAAGAAACAGTCCGACTCAATCTCTTGAATTGAGTCGGACTGTTTCTTA-3′ (SEQ ID NO:18).

Example 5 Quantitative Real-Time PCR

The primer sequences used for human AKT1 were sense:5′-TCTATGGCGCTGAGATTGTG-3′ (SEQ ID NO:19) and antisense:5′-CTTAATGTGCCCGTCCTTGT-3′ (SEQ ID NO:20). Human Actin sense5′-GCTATCCAGGCTGTGCTATC-3′ (SEQ ID NO:21) and antisense:5′-TGTCACGCACGATTTCC-3′ (SEQ ID NO:22). The relative expression levelsof Akt1 transcript was quantified by using the comparative ΔΔC_(t) withActin as an internal control.

Example 6 Immunofluorescence Microscopy

CWR-R1 and 22Rv1 cells were seeded on the cover slides and allowed toattach to the cover slides for overnight. The cells were then fixed withLana's fix for 30 min and washed four times with phosphate-bufferedsaline. The cover slides were blocked in phosphate-buffered salinecontaining 0.3% Triton-X100, 1% bovine serum albumin, and 1% normaldonkey serum 1 hr at room temperature. The newly developed rabbitanti-AR3 antibody was added and incubated for 3 hr at room temperature.After washing with phosphate-buffered saline, the cover slides wereincubated with fluorescein isothiocyanatconjugated anti-rabbit secondaryantibody for 45 mm at room temperature. Finally, the cells werecounterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualizenuclei before mounting. The cover slides were examined by using a Nikondigital microscope system.

Example 7 Immunoprecipitation and Western Blot

The cells were washed twice with ice-cold phosphate-buffered saline andthen lysed with lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mMEDTA, 1 mM EGTA, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 1 mMNa₃VO₄, 1 μg/ml aprotinin, 1 μg/mi leupeptin, and 1 mMphenylmethylsulfonyl fluoride) at 4° C. for 30 mm. The cell lysates werecentrifuged to remove cell debris before incubation with the antibody at4° C. for 1 hr. The immunocomplexes were collected with proteinA-Sepharose beads. The immunoprecipitates were then resuspended in SDSsample buffer and resolved by SDS-polyacrylamide gel electrophoresis.Immunoblotting was performed as described previously (Qiu, Y. et al.,Nature 393, 83-5 (1998)).

For the samples from prostate xenograft tumors, the protein lysates wereprepared by using the tissue protein extraction reagent (Pierce), andthen followed by the same procedures as above. The resultingsupernatants of the cell lysates were mixed with SOS sample buffer andresolved by SDS-polyacrylamide gel electrophoresis. Immunoblotting wasperformed as described. For the prostate tumor samples from mice andpatients, the protein lysates were prepared by using the tissue proteinextraction reagent (Pierce), and then followed by the same procedures asabove.

Example 8 Luciferase Reporter Assay

Luciferase assay was carried out as described previously with minormodifications (Kim, O. et al., Oncogene 23, 1838-44 (2004)). Briefly,cells grown in 24-well plates were transiently transfected withdifferent plasmid constructs using LipofectAMINE 2000 (Invitrogen) forLNCaP cells and FuGENE HI) (Roche) for COS-1 cells following themanufacturer's instruction. At 24 hr post transfection, the cells wereincubated with fresh phenol-red free serum-free medium, or in theexperiments with DHT, with phenol-red free medium containing 5%charcoal-stripped FBS, for 24 hr. Dual-Luciferase assays were performedaccording to the protocol from the manufacturer (Promega). The resultsare presented as the relative promoter activity that is the changes ofluciferase activity relative to the untreated control.

Example 9 Chromatin Immunoprecipitation

CWR-R1 cells were cultured in phenol-red free RPMI 1640 mediumcontaining 5% charcoal-stripped serum for 2 days and then the treatmentwith vehicle or 10 nM DHT for 1 hr. Cells were cross-linked with 1%formaldehyde and then sonicated, the soluble chromatins wereimmunoprecipitated as described previously.

The PCR primers were as follows:

AKT1 P1 ARE, Q377A, sense 5′-CCACAGAGCACCTCAGCAGTCC-3′, (SEQ ID NO: 23)antisense Q377B, 5′-GAGCAGGGCACCCTCTCATGG-3′; (SEQ ID NO: 24) AKT1 P2ARE Q378A, 5′-GCTCCTCACTGACGGACTTGTCTG-3 (SEQ ID NO: 25) and Q378B,5′-CCCCTGGTGACAGATGGCC-3′; (SEQ ID NO: 26) AKT1 P3 ARE Q380A,5′-GTGCATTTGAGAGAAGCCACGCTG-3′ (SEQ ID NO: 33) and Q380B,5′-CACATTGCGCATAGCTGCAGAAG-3′; (SEQ ID NO: 27) PSA Enhancer ARE Guo43A,5′-ACAGACCTACTCTGGAGGAAC-3′; (SEQ ID NO: 28) and Guo43B5′-AAGACAGCAACACCTTTTT-3′. (SEQ ID NO: 29)

Example 10

In vitro cell growth assay and In vivo tumor growth in xenograft modelsLNCaP, CWR-R1 and 22Rv1 cells were infected with the lentivirusesbearing the shRNA sequences and the AR3 expression constructs asindicated. At 48 hr postinfection, the cells were cultured in phenolred-free RPMI1640 medium containing 5% charcoal-stripped serum for 2weeks. The cell colonies were visualized by Coomassie Blue staining. Thetumor growth of LNCaP, 22Rv1 and CWR-R1 in the xenograft models wascarried out as described previously. (Craft, N. et al., Nat Med 5, 280-5(1999); Long, B. J. et al., Cancer Res 60, 663 0-40 (2000)). Briefly, at48 hr postinfection, 10⁶ cells were mixed with 100 μl of Matrigel andthen subcutaneously (s.c.) injected in the left or right flank of thecastrated male SCID/nude mice as indicated in the figure legends. Thevolumes of the tumors formed on both sides were measured weekly andcalculated by using the formula: 0.5236×r₁ ²×r₂ (r₁<r₂). The differencesin the sizes of the tumors formed on both sides were compared by thepaired t-test.

Example 11 Immunohistochemical Analysis

The prostate tissue microarrays used in this study were prepared by NYUCooperative Prostate Cancer Tissue Resource as described previously.(Guo, Z. et al., Cancer Cell 10, 309-19 (2006)). The Vectastain EliteABC Kit (Vector Laboratories) was used for immunohistochemical stainingSections were deparaffinized with xylene and rehydrated through gradedalcohol washes followed by antigen retrieval in a water bath at 98° C.for 45 mm in an antigen unmasking solution (Vector Laboratories). Slideswere incubated in 0.3% hydrogen peroxide to quench endogenoushorseradish peroxidase for 30 min, and then blocked by normal goat serumand subsequently incubated with the anti-AR3 or anti-AR antibody at 4°C. overnight. The slides were then treated with biotin-labeledanti-rabbit IgG and incubated with preformed avidun biotin peroxidasecomplex. Finally, the sections were counterstained with hematoxylin,dehydrated, mounted and examined. The pair-wise group comparison wasconducted by non-parametric Kruskal-Wallis test. Kaplan-Meier analysiswas used for testing the association of AR3 staining and PCA recurrence.The statistical analyses were carried out by using SAS version 9.1software (SAS Institute Inc. Cary, N.C., USA).

Example 12 Microarray Analysis

Total RNA was extracted from CWR-R1 or 22Rv1 cells treated with shAR3-1,shARa and the scrambled shRNA control, respectively. RNA quality andquantity were evaluated using the RNA 6000 Nano kit on AgilentBioanalyzer (Agilent technologies, Palo Alto, Calif.). For each cellline, mRNA from treated cell line and control, labeled with fluorescentdyes, Cy5 and Cy3 respectively, was reverse-transcribed to cDNA andsimultaneously hybridized to an Agilent microarray containing 44k 60mersin the sense orientation. Fluorescent labeling of RNA samples wasperformed according to standard labeling protocols and kits designed forAgilent Human Whole Genome Expression system (Agilent Technologies, PaloAlto, Calif.). Hybridization was carried out using the conditionsspecified in the Agilent Human Whole Genome Expression system. Scannedimages were processed for quality assessment and preprocessing of imagedata using the Agilent Feature Extraction software (AgilentTechnologies). The software quantifies feature signals and theirbackground, performs dye normalization and calculates feature log ratiosand error estimates. The error estimates, based on an extensive errormodel and pixel level statistics calculated from the feature andbackground for each spot, are used to generated a p-value for each logratio. The differential expressed genes were identified by theirp-value<0.05 and a minimal 1.4 fold change in both cell lines treatedwith a given specific shRNA compared to the scrambled control. Thechanges of some identified genes were validated by the quantativereal-time PCR.

Example 13 Overexpression of AR3 Promotes Proliferation in ProstateGland

To better understand the role of human AR3 in prostate cancer, the AR3transgenic (AR3Tg) mouse has been established by using the modifiedprobasin promoter ARR2PB which is positively regulated by androgen andexpressed in the prostate epithelium of sexually mature mice (FIG. 14A).The genotypes of the offspring were determined by tail snipping andfollowed by PCR with a pair of primers specific for human AR3 (FIG.14B). The expression of human AR3 transcript and protein in thetransgenic prostate was confirmed by RT-PCR and Western Blot (FIG. 14C).The transgene positive mice and their negative littermates weresacrificed at 2-month old. The prostate gland was dissected and fixed inFormalin. The serial sections of paraffin-embedded tissues were stainedwith Hematoxylin and Eosin (H & E). Histological analysis of tissuesections revealed that the AR3Tg displayed extensive hyperplasia in allthree lobes (FIG. 15), which was accompanied with an increase inKi67-positive cells (FIG. 16). Interestingly, the number of p63-positivecells is significantly increased in AR3Tg prostates (FIG. 17),suggesting that overexpression of AR3 may promote expansion of basalcells or compromise terminal differentiation of luminal epithelial cell.

Overexpression of AR3 modulates multiple signaling pathways and promotesexpansion of Sca-1+ cell population in mouse prostate.

To delineate the mechanisms by which AR3 exerts its biological activityin prostates, we performed gene microarray analysis using AffymatrixMouse Gene-1.0st-v1 arrays to identify differentially expressed genes inAR3Tg prostates (n=3) in comparison with the age-matched two-month-oldWT littermates (n=3). Our initial analysis revealed that 96 genes weredown-regulated and 138 genes were up-regulated at least 2-fold (withp<0.05) in AR3Tg. The Ingenuity Pathway analysis suggested that AR3 maybe involved in regulation of multiple signaling pathways activated bygrowth factors. This was supported by the elevated MAPK and Akt pathwaysin AR3Tg prostate determined by Western blot with anti-phosphoMAPK andpan anti-phospho AKT-substrates antibodies, respectively (FIG. 18). FIG.19 summarized a partial list of genes regulated by AR3 in mouseprostate. One of the most up-regulated genes is the prohormoneconvertase Pcsk1 which was increased 48 fold in addition to somewell-established AR target genes such as kallikrein family proteins(Klk1 and Klk1b27) and androgen-binding proteins (Abpb and Abpd).Because Pcsk1 is known to process pro-TGF-β, we examined the level ofmature TGF-β in AR3Tg by Western Blot under reducing conditions using apan anti-TGF-β antibody. FIG. 20 shows that the level of mature TGF-βmonomers (doublet suggesting at least two members of TGF-β family may beprocessed in prostate) was dramatically elevated in AR3Tg prostatecompared to the WT control. As a result, both SMAD1 and SMAD3phosphorylation is elevated FIG. 20. Klk1 was reported to be able todegrade IGFBP3 (Rajapakse, S. et al., Mol Reprod Dev, 74: 1053-1063,2007).

Increased Klk1 is expected to free more biological active IGF1 to bindto its receptors. Akt1, previously identified as an AR3 preferred targetin human PCA cells, was modestly but significantly elevated in AR3Tg(FIG. 19). Thus, the elevated Akt activity detected in AR3Tg prostate(FIG. 18) may possibly, at least in part, due to increase of free IGF1level and Akt1 protein level. These changes appear to be consistent withour observation in human PCA cell lines that MAPK, Akt and TGF-βsignaling pathways are altered when AR3 is knocked-down (Guo, Z., etal., Cancer Res, 69: 2305-2313, 2009).

Interestingly, two highly related androgen-binding protein genes Abpb(down 100 fold) and Abpd (up 96 fold) located closely on Chromosome 7were differentially regulated by AR3, suggesting that the effects of AR3on its target genes are dependent on promoter and/or cell context. Mostintriguingly, a modest but significant increase of a set of genesassociated with stem/progenitor cells was detected in AR3Tg includingLy6a/Sca-1, Kit, aldehyde dehydrogenase (Aldh1a1), Notch1, Jag1,Frizzled homolog 6 (Fzd6) (FIG. 19). To determine expression of Sca-1and α6 integrin (CD49f), prostates from 5-week-old AR3Tg or WTlittermates were dissected and digested in 0.5% collagenase. Thedissociated prostate cells were stained with FITC-anti-Sca-1 andPE-anti-CD49f antibodies. Fluorescence-activated cell sorting (FACS)analysis of double-stained cells was performed on a FACSCalibur flowcytometer, using vendor-provided CellQuest software. FIG. 21 shows thatthe number of Sca-1 and α6 integrin double positive cells was increasedat least two fold to 35% in AR3Tg prostates compared to 16% in the WTlittermate controls, suggesting that the stem/progenitor cell populationmay expand in AR3Tg prostates. To test whether the AR3 Tg prostatecontains more stem/progenitor cells that is capable to generate spheresfrom a single cell, we further performed prostatic sphere formation inMatriGel as described previously (Xin, L., et al., Stem Cells, 25:2760-2769, 2007). FIG. 22 shows that the prostatic cells from the AR3Tgappeared to form more spheres than those from the WT littermate controlsand AR3 transgene is highly expressed in the basal compartment of thesphere. These data are consistent with our observation in human prostatetissues and suggest that AR3 plays a role in regulating prostatestem/progenitor activity.

Our IHC screening performed on multi-tumor tissues arrays revealed thatAR3 appeared to be detected in many types of tumors (FIG. 23), includingTestis Seminom, Testis Germ cell tumor, Lung Carcinoid tumor, LungAdenocarcinoma, Breast Ductal Carcinoma, Breast Invasive LobularCarcinoma, Basal Cell Carcinoma and Pancreas Adenocarcinoma.

Association of AR and AR Splicing Isoforms with Mitochondria

When we overexpressed AR4 in COS-1 cells and performedimmunofluorescence staining with anti-AR (N-20), AR4 (green) isprimarily detected in punctuate structures in the cytoplasm, which arelabeled by the mitotracker (red) (FIG. 24A), suggesting that AR4 may bepresent in mitochondria instead of nuclei. This possibility is furthersupported by the fractionation experiments shown in FIG. 24B. Itappeared that a portion of AR and AR splicing variants are detected inthe mitochondria fraction isolated by using the Mitochondria Isolationkit (Pierce). The detected bands were diminished in cells treated with acocktail of shRNAs for AR Exon 1 and Exon 5, suggesting that these bandsare indeed AR and AR isoforms.

Example 14 Cloning and Characterization of AR8

We have cloned a novel AR splicing variant AR8 (FIG. 25A) and itsnucleotide sequence is shown in FIG. 25B. AR8 contains the NTD and a33-a.a unique sequence derived from a cryptic exon 2b located in theIntron 2 (FIGS. 25A & 26A). When we overexpressed AR8 in COS-1 cells andperformed immunofluorescence staining with anti-AR (N-20), AR8 isprimarily present on the plasma membrane (FIG. 26B). We have identifiedtwo Cysteine residues (C558 & 569) in the C-terminal unique sequence aspotential palmitoylation sites (underlined in FIG. 26A). Substitution ofthese two Cys with Ala residues dramatically diminished plasma membranetargeting of AR8 (FIG. 26B). The real-time PCR analysis revealed thatAR8 expression is elevated in hormone resistant CWR22R xenografts (HR)compared to hormone sensitive counterpart (HS) (FIG. 27A) and AR8 levelis also increased in hormone resistant LNCaP derivatives C4-2 and C4-2Bcompared to hormone sensitive parental LNCaP cells (FIG. 27B). When wetreated hormone-resistance prostate cancer cell lines C4-2, 22Rv1 andCWR-R1 with the lentivirus encoding the shRNA specific for AR8 (targetsequence: CTCATTATCAGGTCTATCA) (SEQ ID NO:30), growth of these cells wasattenuated (FIG. 28A). This is accompanied with the reduced activity ofAkt, MAPK and Src kinases (FIG. 28B). We further examined the effects ofAR8 knock-down on cell proliferation and apoptosis as describedpreviously. We were able to show that AR knock-down led to inhibition ofproliferation (FIG. 29A) and increase of apoptosis in CWR-R1 cells (FIG.29B).

TABLE 1 Genes commonly regulated by AR3 and AR Gene Change upon symbolBiological process knockdown STK32B cell signaling up SYT4 vesiculartrafficking and exocytosis CPA1 proteolysis, zymogen inhibition GIPRsecretion of insulin, anabolic response GPR101 cell signaling MAGEA10embryonic development, tumor transfor- mation and progression OTORcartilage development and maintenance ONECUT1 transcriptional activator,liver gene transcription IFNA10 inflammatory response ZNF224transcriptional regulation NBN cellular response to DNA damage ITTH5hyaluronan metabolic process ZNF624 transcriptional regulation DYNLT3motor activity B4GALT4 cell metabolism IGFBP3 growth factor signalingPNPLA4 lipid metabolism PTPRK regulation of processes involving cellcontact and adhesion ANKRA2 cell endocytosis PCDH10 cell adhesion,mesoderm development CD19 B cell receptor signaling pathway CDH18 celladhesion ZNF589 DNA binding protein, transcription down regulationANGPTL4 inhibiting vascular activity as well as tumor cell motility andinvasiveness PDXP coenzyme for biochemical homeostasis CSRP1 neuronaldevelopment PPP5C RNA biogenesis and/or mitosis TRAF4 adapter proteinand signal transducer NT5DC3 lipid metabolism PPFIA3 disassembly offocal adhesions SRF MAPK signaling pathway GAS1 tumor suppressor geneLDHA pyruvate metabolism CHRNA2 modulation of ion-conducting channelsTMEM81 transmembrane cell component SLC25A37 protein transportationUBE2M protein-ubiquitination pathway DHRS2 reactive carbonyls metabolismSNRPB pre-mRNA splicing or in snRNP structure TPM1 striated musclecontraction SNAP23 transport vesicle docking and fusion FKBP5 proteinbinding, immunoregulation PRKAR2A protein transport HNRPA0 pre-mRNAsplicing or in snRNP structure TMED9 protein transportation andlocalization

TABLE 2 Genes preferentially regulated by AR Gene Change upon symbolBiological process knockdown PCDH11Y cell-cell recognition up HOXA13transcription factor, involved in embryonic development CDKN1A cellcycle progression PCDH9 neuronal connections and signal transduction FASapoptosis MYBL1 proto-oncogene, transcription activator TAF9BDNA-binding, transcription regulation AR transcription factor down CLUinhibit apoptosis WNK3 serine-threonine protein kinase, cell signalingTCF3 transcription factor in cell differentiation IL6ST cytokine signaltransduction (gp130) TERT oncogenesis cellular senescence DACH2transcriptional cofactor TMEPAI Androgen induced gene KLK3 serineproteases, biomarker for prostate cancer KLK15 serine proteases, cancerbiomarker CLDN4 component of cell tight junctions CDC2 cell cycleregulation WNT3 embryogenesis WNT10B Inhibition of adipogenesis

TABLE 3 Genes preferentially regulated by AR3 Gene Change upon symbolBiological process knockdown EPHA3 Ephrin receptor, cell signaling upAF268194 cell signaling (IRA2) Neuralin-1 BMP (bone morphogeneticprotein) pathway OPTN TNF-alpha signaling pathway RYR2 Cardiac muscleryanodine receptor SCML2 transcription repression RFX3 transcriptionfactor AI278811 transcription factor, proto-oncogene (MYC like) EFCBP2EF-band calcium binding protein 2 TES Zinc finger ion binding proteinSLC40A1 solute carrier, iron-regulated transporter SLC7A11 amino acidtransport RGMB cell membrane lipid-anchor SYTL2 vesicle traffickingAPOLD1 angiogenesis DMD actin binding, cytoskeletal anchoring NTNG1neurite outgrowth GLUD1 nitrogen metabolism AKT1 Proto-oncogene,Serine/threonine-kinase down AKT1S1 Akt-mTOR pathway WNK1Serine/threonine-kinase, signal transduction MAP4K4 MAP Kinase PathwaysADCY6 signal transduction PICK1 organize subcellular localization ofmembrane proteins HDAC3 chromatin modification, epigenetic SELENBP1Golgi protein transport EVI5 regulator of cell cycle progression ELK1Ets family transcriptional factor, proto- oncogene SLC2A4RGtranscription factor ARFGAP1 membrane trafficking and vesicle transportPRG2 immune response PLEKHA3 lipid binding DNM2 receptor mediatedendocytosis MDM4 Proto-oncogene, p53 binding protein RAP1GAP GTPaseactivity, cell signaling CD2BP2 mRNP assembly FXR2 RNA binding SCUBE1adhesive molecule TPM1 striated muscle contraction AP3M1 vesiclestrafficking KDELR2 endoplasmic reticulum protein trafficking RAB6IP1Rab6-mediated GTPase signaling TSR2 signal transduction GYS1 glycogensynthase 1, glucose metabolism LIMD1 tumor-suppressor gene ARNTL2circadian rhythms EMD nuclear envelope assembling METTL7Amethyltransferase, chromatin modification HOXB7 transcription factor,inhibition of differentiation MDFI transcription repressor, inhibitionof differentiation TAF9B DNA binding, transcription regulation

1. An isolated nucleic acid molecule comprising a polynucleotideselected from the group consisting of: (a) a human androgen receptorsplice variant encoding AR3 polypeptide (SEQ ID NO: 5); (b) a humanandrogen receptor splice variant encoding AR4 polypeptide (SEQ ID NO:6);(c) a human androgen receptor splice variant encoding AR5 polypeptide(SEQ ID NO:7); (d) a human androgen receptor splice variant encoding AR8polypeptide (SEQ ID NO:8); (e) a human androgen receptor splice variantencoding AR4b polypeptide (SEQ ID NO:37); (f) a polynucleotide sequencecomplementary to the polynucleotide sequence of (a), (b), (c), (d) or(e); and (g) polynucleotide sequence that is at least 90% identical tothe polynucleotide sequence of (a), (b), (c), (d), (e) or (f).
 2. Anisolated androgen receptor polypeptide selected from the groupconsisting of: (a) a human androgen receptor splice variant AR3polypeptide (SEQ ID NO: 5); (b) a human androgen receptor splice variantAR4 polypeptide (SEQ ID NO:6); (c) a human androgen receptor splicevariant AR5 polypeptide (SEQ ID NO:7); (d) a human androgen receptorsplice variant AR8 polypeptide (SEQ ID NO: 8); (e) a human androgenreceptor splice variant AR4b polypeptide (SEQ ID NO: 37); (f) apolypeptide having a sequence that is at least 90% identical to thepolypeptide sequence of (a), (b), (c), (d) or (e).
 3. A pharmaceuticalcomposition comprising a therapeutically effective agent which regulatesexpression of an androgen receptor splice variant from claim
 1. 4. Theisolated nucleic acid of claim 1 or a fragment thereof, wherein saidnucleic acid is a prognostic marker for prostate cancer.
 5. An isolatedantibody that recognizes an epitope on a human androgen receptor splicevariant polypeptide of claim
 2. 6. The isolated nucleic acid molecule ofclaim 1, wherein said nucleic acid is in a kit.
 7. The isolated nucleicacid molecule of claim 1, wherein said nucleic acid is that of part (a).8. The isolated nucleic acid molecule of claim 1, wherein said nucleicacid is that of part (b).
 9. The isolated nucleic acid molecule of claim1, wherein said nucleic acid is that of part (c).
 10. The isolatednucleic acid molecule of claim 1, wherein said nucleic acid is that ofpart (d).
 11. The isolated nucleic acid molecule of claim 1, whereinsaid nucleic acid is that of part (e).
 12. The isolated nucleic acidmolecule of claim 1, wherein said nucleic acid is that of part (g). 13.A method for inhibiting the function of an androgen receptor splicevariant in a prostate cancer cell, comprising delivering an agent to thecell selected from the group consisting of an inhibitor of transcriptionof the androgen receptor splice variant, an inhibitor of translation ofthe androgen receptor splice variant, and an antagonist of the androgenreceptor splice variant, wherein the androgen receptor splice variant isselected from the group consisting of AR3, AR4, AR4b, AR5 and AR8. 14.The method of claim 13, wherein the agent is an inhibitor of translationof the androgen receptor splice variant and decreases the stability ofthe mRNA.
 15. The method of claim 13, wherein the agent is an inhibitorof transcription of the androgen receptor splice variant.
 16. The methodof claim 14, wherein the agent is selected from the group consisting ofantisense technology, RNA inhibition technology (RNAi), and ribozymes.17. The method of claim 13, wherein the agent is an isolated antisensenucleic acid that is sufficiently complimentary to the nucleic acidsequence of SEQ ID NOS: 1, 2, 3 or 4 to permit hybridization underphysiologic conditions, and which antisense nucleic acid inhibitsexpression of the androgen receptor splice variant.
 18. The method ofclaim 13, wherein the agent is a siRNA and is directed against SEQ IDNO:30 of AR8.
 19. The method of claim 13, wherein the agent is anantagonist of the androgen receptor splice variant polypeptide.
 20. Themethod of claim 13, wherein the agent is an inhibitor of transcriptionof the androgen receptor splice variant and degrades transcriptionfactors required for androgen receptor splice variant transcription.